Chlamydia trachomatis is an obligate intracellular bacterial pathogen responsible for human genital and ocular infections. Species of Chlamydia utilize a type-III secretion system to deliver bacterial effector proteins into the host cell in order to promote invasion and establish residence within a parasitophorous vacuole called an inclusion. The effector protein Tarp has been previously implicated as an important effector for promoting invasion during Chlamydia trachomatis infection by directing the formation of new actin filaments and bundles. Intriguingly, the significance of Tarp mediated cytoskeletal changes has not been fully explored in vivo. Host-pathogen interaction studies that replicate the human infection can be performed with mouse adapted Chlamydia, Chlamydia muridarum. However, the genetic tools to create gene deletions in C. muridarum have been lacking. Recently, our collaborators in the Fields and Wolf Laboratories developed a novel genetic tool for creating Tarp deletion mutants and complement clones in Chlamydia muridarum. Through the use of this tool, we were able to study the significance of Tarp in a murine infection model. In addition to Tarp, two other early effectors TmeA and TmeB are hypothesized to play a role in invasion, but a full account of their involvement remained unknown. In our studies, we were able to determine the roles of TmeA and TmeB in remodeling the host cytoskeleton. Using biochemical crosslinking assays, and actin polymerization studies, we discovered that TmeA has the ability to activate host protein N-Wasp in order to increase Arp2/3-dependent actin polymerization, while TmeB can in turn inhibit Arp2/3-directed actin polymerization via direct interactions with Arp2/3. Collectively, these are important findings as our studies have revealed how a collection of early chlamydial effectors work to modulate the host cytoskeleton to facilitate Chlamydia infections.

El objetivo general de esta tesis fue avanzar en el estudio de la relación patógeno-hospedador-ecosistema de la infección por C. trachomatis en mujeres de la ciudad de Bahía Blanca, para poder favorecer medidas de control y prevención eficaces. Las poblaciones estudiadas fueron mujeres sintomáticas y asintomáticas atendidas en un hospital público (n=295), trabajadoras sexuales (n=143) y jóvenes ingresantes universitarios de ambos sexos (n=204). La prevalencia de infección por C. trachomatis fue mayor en las trabajadoras sexuales que en las estudiantes universitarias (6,29 % y 5,88 % respectivamente). La menor prevalencia se observó en las mujeres sintomáticas y asintomáticas atendidas en un hospital público (3,05 %). En este grupo, la frecuencia de infección fue mayor en las mujeres asintomáticas (4,35 %) que en las sintomáticas (2,65 %). Entre las muestras positivas para C. trachomatis el genotipo E fue el de mayor circulación, seguido por el D y el F. La presencia de C. trachomatis se asoció significativamente con la menor edad, el mayor número de parejas sexuales, el cambio reciente de pareja, la alteración de la microbiota habitual vaginal y la presencia de reacción inflamatoria vaginal. En las trabajadoras sexuales y en las mujeres sintomáticas y asintomáticas, la vaginosis bacteriana fue el estado patológico más frecuente y la trichomonosis la ITS más prevalente. La frecuencia de otras infecciones genitales como candidiasis y sífilis fue baja. No se detectaron casos de infección por N. gonorrhoeae ni serología positiva para HIV. En las jóvenes universitarias, los factores de riesgo para la adquisición de ITS fueron el inicio temprano de las relaciones sexuales, la baja utilización del preservativo y la falta de consulta a los profesionales especializados, los cuales alertan sobre la necesidad de incrementar la vigilancia en esta población. Los resultados muestran la necesidad de profundizar el estudio de las infecciones genitales, y en particular las causadas por C. trachomatis, en las mujeres jóvenes, independientemente de la sintomatología, y encarar programas de prevención de ITS, especialmente en las poblaciones más vulnerables. Palabras clave: Chlamydia trachomatis, ITS, trichomonosis, vaginosis bacteriana, candidiasis, sífilis, HIV, trabajadoras sexuales, jóvenes universitarios. / The general aim of this thesis was to advance in the study of the pathogen-host-ecosystem relation of infection by C. trachomatis in women of the city of Bahía Blanca, to promote effective control and prevention measures. The studied populations were symptomatic and asymptomatic women treated in a public hospital (n=295), female sex workers (n=143) and young college students of both sex (n=204). The prevalence of infection by C. trachomatis was higher in female sex workers than in college students (6.29% y 5.88 % respectively). The lower prevalence was observed in symptomatic and asymptomatic women treated in a public hospital (3.05%). In this group, the frequency of infection was higher in asymptomatic women (4.35%) than in the symptomatic women (2.65%). Between the positives samples for C. trachomatis, the E genotype was the one with most circulation, followed by genotypes D and F. The presence of C. trachomatis was significantly associated with lower ages, bigger number of sexual partners, therecent change of partner, the alteration of the normal vaginal microbiota and the presence of vaginal inflammatory reaction. In female sex workers, and in symptomatic and asymptomatic women, the bacterial vaginosis was the most frequent pathological state and the trichomonosis was the most prevalent STI. The frequency of other genital infections such as candidiasis and syphilis was low. There were no cases detected of infection by N. gonorrhoeae or positive serology for HIV. In young college students, the risks factors for the acquisition of STI were the early beginning of sexual intercourse, low utilization of condom and the lack of consultation to specialized professional, which alert about the need of increase the surveillance in this population. The results show the need of deepen the study of genital infections, and particularly the ones caused by C. trachomatis, in young women, independently of the symptomatology, and encourage STI prevention programs, especially in the most vulnerable populations. Key words: Chlamydia trachomatis, STI, trichomonosis, bacterial vaginosis, candidiasis, syphilis, HIV, female sex workers, young college students.

Background: Selected factors place Dominican female adolescent and adults at risk for sexually transmitted infections (STIs) such as Chlamydia, causative organism Chlamydia trachomatis, and Gonorrhea, causative organism Neisseria gonorrhoeae. The purpose of this study was to determine the prevalence of Chlamydia and Gonorrhea among adolescent and adult females that utilize the clinic in Veron, Dominican Republic. Clinical standards of care for these STIs and educational programs for prevention were developed from the data gathered from this study. Significance at 0.05 ά of the relationship of educational level, management of risk factors and other selected independent variables on prevalence rate of Chlamydia and Gonorrhea in the clinic population of Veron, Dominican Republic were determined. The objectives of the study were to 1) determine the prevalence of adolescent and young adult females diagnosed with Gonorrhea and Chlamydia who visit the clinic for prenatal visits, annual pap smear exams and gynecological complaints; 2) determine the extent to which educational level is a predictor of positive diagnosis or risk for infection of Chlamydia and Gonorrhea and; 3) determine which selected demographic and risk factors are associated with positive test results for Gonorrhea and Chlamydia. Methods: All adolescent and adult females ages 15 years and older visiting the clinic in Veron for prenatal exams, pap smear exams and gynecological complaints between January 28, 2008 – March 3, 2008 were invited to participate in this prevalence study. Of the 90 invited, the accepting sample was 90 who signed an informed consent form. Prior to STI testing each patient completed a verbal interview and questionnaire on sociodemographic characteristics as well as knowledge, attitudes, and beliefs related to Chlamydia and gonorrhea, sexual experiences and behaviors and illicit drugs use. Specimens collected from the endocervical canal of each female were tested and results provided within two hours, followed by immediate treatment by a licensed Dominican physician and follow-up care based on the guidelines and standards of care. The data were analyzed using descriptive statistics, chi square, t-test and logistic regression. Results: A total of ninety women participated in the study. Chlamydia was detected in 6.7% of the patient population and Gonorrhea was detected in 22.2% of the patient population. Co-infection of both Chlamydia and Gonorrhea was present in 2 cases. Among the positive Chlamydia tests results, 50% had less than a six-year education and 50% had more then six years of education. In addition, 83.3% of the patients with positive Chlamydia results answered "yes", they could read and 16.6% stated they could not read, while 83.3% of the patients with positive Chlamydia results stated they could write and 16.6% stated they could not write (P>0.05). Among the patients that tested positive for Gonorrhea, 55% stated they had less than six years of formal education and 45% had more than six years of formal education (P>0.05). There were 75% of the patients that tested positive for Gonorrhea that stated they could read and 25% who stated they could not read (P>0.05); while 85% of the patients with positive Gonorrhea results stated they could write and 15% stated they could not write (P> 0.05). Conclusion: Educational level and other selected demographic characteristics and risk factors in this study are not a significant predictor of positive diagnosis or risk of infection for Chlamydia or Gonorrhea. We cannot conclude that specific risk factors are associated with positive test results for Gonorrhea and Chlamydia. For the physicians involved in the clinical decision-making regarding the female patients at the Veron clinic, more data are needed to determine appropriate populations for screening of Gonorrhea and Chlamydia as well as appropriate educational tools on sexually transmitted infections. / Ph. D.

Folate is an essential cofactor required for several processes including DNA and amino acid biosynthesis. Folate molecules are made up of three parts: a pteridine ring, p-aminobenzoate (pABA), and a variable number of glutamate residues. Chlamydia trachomatis synthesizes folate de novo; however, several genes encoding enzymes required for the canonical folate biosynthesis pathway are missing, including pabA/B and pabC, which are normally required for pABA biosynthesis from chorismate. Previous studies have found that a single gene in C. trachomatis, CT610, functionally replaces the canonical pABA biosynthesis genes. Interestingly, CT610 does not use chorismate as a substrate. Instead, the CT610-route for pABA biosynthesis incorporates isotopically labeled tyrosine into the synthesized pABA molecule. However, in vitro experiments revealed that CT610 produces pABA without any added substrates (including tyrosine) in the presence of a reducing agent and molecular oxygen. CT610 shares low sequence similarity to non-heme diiron oxygenases and the previously solved crystal structure revealed a diiron active site. Taken together, CT610 is proposed to be a novel self-sacrificing enzyme that uses one of its active site tyrosine residues as a precursor to pABA in a reaction that requires O2 and a reduced metallocofactor. Here, we discuss our progress towards understanding CT610-catalyzed pABA synthesis. Upon investigation of the pABA production and oxygenase activities of several active site tyrosine to phenylalanine variants, we found that Y27 and/or Y43 are the most likely precursors to the resulting pABA molecule. Further, activity was nearly completely abolished with a K152R variant, suggesting that this conserved lysine may be the required amino group donor. We also developed an in vitro Fe(II) reconstitution procedure, where the reconstituted enzyme exhibited a drastic increase in oxygenase activity but, surprisingly, a significant decrease in pABA synthase activity. Interestingly, a significant increase in pABA synthase activity was observed when the enzyme was reconstituted with manganese as opposed to iron, suggesting that the diiron active site of this enzyme might not be directly involved in CT610-dependent production of pABA and instead Mn may be the actual cofactor. Finally, we show that two 18O atoms from molecular oxygen are incorporated into the pABA molecule when synthesized by Mn-reconstituted CT610, providing further evidence for the oxygenase activity of CT610 and supporting our proposed mechanism that involves two monooxygenase reactions. / Master of Science in Life Sciences / Folate is an essential molecule that is required for all cells to survive. Folate is usually made in the cell with the help from proteins known as enzymes. Enzymes help biochemical reactions happen by speeding up the rate of their specific chemical reaction. In order for this to occur, an enzyme binds to a very specific molecule, called a substrate, and facilitates the reaction transforming the substrate into a new product while not altering the enzyme in the process, allowing for the protein to continuously facilitate this reaction. Chlamydia trachomatis is the strain of bacteria that causes one of the most common sexually transmitted infections in the US, Chlamydia. These bacteria make folate themselves but have been shown to make this molecule in a very different way from an average folate-synthesizing organism. One enzyme in C. trachomatis known as CT610 has been shown to participate in this unusual route to produce folate. Interestingly, CT610 is thought to remove part of itself to donate to the molecule it produces, effectively killing the enzyme after only one reaction. In this study we show that CT610 performs very unique chemistry to ultimately facilitate the production of folate to allow C. trachomatis to survive. This knowledge could be used in the future for the design of antibiotics specifically targeting C. trachomatis and thus treating the infections caused by this organism.

Tetrahydrofolate (THF) is an essential cofactor for one-carbon transfer reactions in various biochemical pathways including DNA and amino acid biosynthesis. This cofactor is made up of three distinct moieties: a pteridine ring, p-aminobenzoate (pABA), and glutamate residues. Most bacteria and plants can synthesize folate de novo, unlike animals that obtain folate from their diet. An established pathway for THF biosynthesis exists in most bacteria, but there is evidence of some organisms such as Chlamydia trachomatis and Nitrosomonas europaea which do not contain the canonical THF biosynthesis genes, despite still being able to synthesize THF de novo. Previous studies have shown that these organisms do not contain the pabABC genes, normally required to synthesize the pABA portion of THF, and can circumvent their presence with just a single gene: ct610 and ne1434 from C. trachomatis and N. europaea, respectively. Interestingly, these novel enzymes for pABA synthesis do not use the canonical substrates, chorismate or other shikimate pathway intermediates. The gene product of ct610 was named Chlamydia Protein Associating with Death Domains (CADD) due to its established role in host mediated apoptosis, while the crystal structure showed an architecture similar to know diiron oxygenases. However, we provide evidence of a moonlighting function in pABA synthesis. Isotopic labeling experiments to understand what substrate might be used by CADD found that isotopically labeled tyrosine was incorporated into the final pABA product. Compellingly, CADD was able to produce pABA in the presence of molecular oxygen and a reducing agent alone without the addition of any exogenous substrate, implicating this unusual enzyme as a self-sacrificing pABA synthase from C. trachomatis. Here, we provide strong evidence for Tyr27 being a sacrificial residue that is cleaved from the protein backbone to serve as the pABA scaffold. Furthermore, we also provide evidence that K152 is an internal amino donor for this pABA synthase reaction performed by CADD. In the case of NE1434, we have conducted initial experiments such as site-directed mutagenesis and our findings suggest that these self-sacrificing residues are conserved between two distantly related organisms. Finally, the pABA synthase activity is reliant on an oxygenated dimetal cofactor and despite the crystal structure of CADD depicting a diiron active site, we have demonstrated that CADD's pABA synthase activity is dependent on a heterodinuclear Mn/Fe cofactor. Conversely, NE1434 demonstrates no preference for manganese and likely employs a more traditional Fe/Fe cofactor for catalysis. Our results implicate the CADD and NE1434 as self-sacrificing pABA synthases that have diverging metal requirements for catalysis. / Master of Science in Life Sciences / Folate is a molecule used by all organisms that is necessary for survival. Many kinds of bacteria are able to make this molecule with proteins called enzymes, which help by quickening the rate of a reaction. Enzymes are catalysts that usually work by binding a molecule, called a substrate, and will act on this substrate to generate a product; the enzyme remains unchanged in this process, which allows it to facilitate many more of these reactions. Chlamydia trachomatis, which is a leading cause of sexually transmitted infections (STIs) in the United States, and Nitrosomonas europaea, an environmental bacterium, are able to use enzymes to make their own folate, but not in the way that many other bacteria do. These organisms contain enzymes that use a part of their own structure as a substrate, making them "sacrificial lambs". Our study provides evidence of how these organisms carry out an abnormal chemical reaction to make folate which can help scientists target this pathway for the development of antibiotics.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal. / Le cancer du col utérin est la troisième néoplasie en importance chez la femme mondialement. Plusieurs études cliniques et épidémiologiques démontrent que le virus du papillome humain (HP V) est un agent étiologique impliqué, nécessaire au développement des néoplasies du col. Par contre, de plus en plus d'évidences suggèrent une interaction de multiples cofacteurs dans la progression vers le cancer. Nous avons démontré précédemment que des cellules cotransfectées par HPV-16 et la séquence Xho-2 du virus herpès simplex type 2 (HSV-2) induisaient des tumeurs dans des souris athymiques, contrairement à des cellules transfectées par HPV-16 ou HSV-2 seuls. Nous nous proposons d'étudier dans cette étude transversale le rôle de HSV-2, ainsi que de Chlamydîa trachomatis (CT) et du virus herpès humain type 6 (HHV-6) comme cofacteurs aux HPV dans la progression des lésions néoplasiques du col. Une cohorte de femmes a été recrutée consécutivement dans une clinique de colposcopie. Des cellules exfoliées du col ont été soumises à des tests PCR standardisés pour la présence de CT et de HHV-6. 27 génotypes de HPV ont été identifiés avec le Line Blot assay. La région transformante Bgl IIC de HSV-2 a été détectée par PCR. La région Xho-2 de HSV-2 a été détectée par une technique de détection par PCR déjà bien établie (PCR Xho-2) auparavant. Parmi les 439 femmes recrutées, 244 étaient normales, 80 avaient des lésions du col de bas grade (LSIL), 65 avaient des lésions de haut grade (HSIL) et 50 avaient im cancer du col. Des HP V à haut risque étaient détectées dans 93.3% des femmes avec HSIL et 20% des femmes normales. HPV-16 était le plus fréquemment détectés parmi les génotypes de HPV. Tous les spécimens étaient négatifs pour les régions transformantes Xho-2 et Bgl IIC de HSV-2. HHV-6 a été détecté dans 10 spécimens toutes lésions confondues. 3 spécimens de femmes avec un cancer étaient positifs pour CT, contrairement à aucun des spécimens de femmes sans lésions (p<0.031). En conclusion, notre étude a démontré que même si la séquence Xho-2 de HSV-2 transformes des cellules in vitro, elle n'a pas été détectée chez des femmes avec des lésions de haut grade du col. Cependant, C. trachomatis et HHV-6 ont été associés significativement avec les lésions de haut grade et cancer du col et pourraient être des cofacteurs aux HPV.

L'objectif principal de cette étude était d'évaluer la performance clinique du test PATH-GC CHECK (Program for Appropriate Technology in Health (PATH), Seattle, USA), un test rapide immunochromatographique pour la détection de la gonococcie génitale. Cette évaluation a été initiée par l'Organisation Mondiale de la Santé (OMS) dans le cadre de la recherche de tests rapides pouvant être utilisés comme alternatives aux méthodes conventionnelles difficiles à pratiquer dans les pays à ressources logistiques et financières limitées. Dans le cadre de ce mémoire, les prévalences de Neisseria gonorrhoeae (NG) et Chlamydia trachomatis (CT) ont été estimées et leurs facteurs de risque étudiés. L'étude a été conduite chez les travailleuses du sexe (TS) de Cotonou et Porto-Novo au Bénin d'octobre 2003 à juillet 2004. Au total, 1133 sujets, répartis en 906 (80%) à Cotonou et 227 (20%) à Porto-Novo, ont été recrutés. Des prélèvements au col de l'utérus et au vagin ont été testés pour NG et CT. La prévalence de NG était de 4,6% (4,2% à Cotonou et 6,3% à Porto-Novo). Celle de CT était de 4,7% (5,3% à Cotonou et 2,4% à Porto-Novo). Le test rapide réalisé sur les prélèvements cervicaux avait une sensibilité (Se) de 70,0% avec un IC à 95% [55,4 -82,1], et une spécificité (Sp) de 97,2% avec un IC à 95% [96,0-98,1]. Ses valeurs prédictive positive (VPP) et négative (VPN) étaient respectivement de 54,7% et 98,5%. Sur les prélèvements vaginaux, les paramètres correspondants étaient de 54,1%, 98,2%, 60,6%, et 97,7% respectivement pour les Se, Sp, VPP et VPN. Cette évaluation peut être, jugée comme concluante étant donné que l'hypothèse de validité de départ pour le contexte de notre étude était une sensibilité d'au moins 50% avec une précision de l'estimation de la sensibilité de plus ou moins 10%. Les seuls facteurs de risque significativement associés à la cervicite à NG et/ou CT étaient d'ordre clinique: les pertes cervicales (RC=5,1 IC à 95% [1,70-15,16]) et le saignement franc après prélèvement au col (RC=2,7 IC à 95% [1,45-5,05]). Ceci confirme l'utilité des algorithmes cliniques de diagnostic de ces infections chez les TS utilisés au Bénin qui basent le diagnostic et le traitement des cervicites sur ces signes cliniques.

Introdução: Chlamydia trachomatis (CT) é causa da infecção sexualmente transmitida de origem bacteriana mais comum. Na mulher, a infecção genital pela CT pode causar cervicite, uretrite, endometrite, salpingite. Infecções persistentes ou recorrentes provavelmente representam um importante fator de risco para o desenvolvimento de sequelas associadas, como dor pélvica crônica, gravidez ectópica e infertilidade por fator tubário. A lectina ligadora de manose (MBL), componente doo sistema imune inato, tem importante papel na defesa antimicrobiana, reconhecendo vírus, fungos e patógenos bacterianos. O gene que codifica para a MBL é polimórfico, e a substituição de um único nucleotídeo resulta na produção de uma proteína instável, que é rapidamente degradada. Objetivo: Avaliar se mulheres brasileiras portadoras de um polimorfismo do gene da MBL apresentam diferentes susceptibilidades para a ocorrência de obstrução tubária, na presença ou ausência de uma infecção prévia por Chlamydia trachomatis. Métodos: Em estudo caso-controle, foram avaliadas 75 pacientes com obstrução tubária e 75 pacientes com tubas pérvias, atendidas na Divisão de Ginecologia do Hospital das Clínicas da Universidade de São Paulo (HCFMUSP). Anticorpos IgG anti-CT foram mensurados através de um ensaio de imunoabsorção enzimática para investigar uma infecção prévia pela CT. Para o polimorfismo, realizou-se coleta de células bucais e o DNA extraído foi analisado através de reação em cadeia da polimerase (PCR), digestão de endonuclease e gel de eletroforese, utilizando pares de primers específicos para a região polimórfica. Todo material foi mantido a -80°C e enviado em gelo seco para a Division of Immunology and Infectious Diseases da Weill Cornell Medicine em Nova York. Associações entre genótipos de MBL ou alelos e permeabilidade tubária foram analisadas pelo teste de qui-quadrado de Pearson com ou sem correção de Yates. Resultados: Não houve diferença na detecção de anticorpos da CT entre os grupos. Mulheres com tubas obstruídas tiveram uma prevalência maior do genótipo AB (36%) versus (16%), resultado estatisticamente significativo (p < 0,01). De maneira semelhante, a distribuição do alelo A e do alelo variante B também apresentaram diferenças significantes entre os grupos (p < 0,01). Conclusão: Os achados sugerem, que embora a exposição à Chlamydia trachomatis tenha sido semelhante em ambos os grupos, a presença do alelo variante B do gene que codifica para a MBL aumenta o risco de desenvolvimento da obstrução tubária, subsequente à infecção pela CT ou outros agentes infecciosos. Nas mulheres brasileiras avaliadas a presença de tal polimorfismo genético aumentou a probabilidade de obstrução tubária em consequência de uma infecção do trato genital / Introduction: Chlamydia trachomatis (CT) is the cause of the most common bacterial sexually transmitted infection. In women, genital CT infection may cause cervicitis, urethritis, endometritis, salpingitis. Persistent or recurrent infections probably represent an important risk factor for the development of associated sequelae, such as chronic pelvic pain, ectopic pregnancy and tubal factor infertility. Mannose-binding lectin (MBL), a component of the innate immune system, has an important role in antimicrobial defense, recognizing viral, bacterial and fungal pathogens. The gene coding for MBL is polymorphic and a single nucleotide substitution results in production of an unstable protein, that is rapidly degraded. Objective: To evaluate whether Brazilian women with a polymorphism in the MBL gene present different susceptibilities to the occurrence of fallopian tube damage, in the presence or absence of a previous infection by CT. Method: In a case-control study, 75 patients with tubal obstruction and 75 patients with patent tubes were studied, all seen at the Gynecology Division of the Hospital das Clínicas of the University of São Paulo (HCFMUSP). IgG anti-CT antibodies were measured by enzyme-linked immunoassay to investigate a previous CT infection. For the polymorphism analysis, buccal cells were collected and the extracted DNA was analyzed by polymerase chain reaction (PCR), endonuclease digestion and gel electrophoresis using primer pairs specific for the polymorphic region. All material was maintained at -80 ° C and sent on dry ice to the Division of Immunology and Infectious Diseases at Weill Cornell Medicine in New York. Associations between MBL genotypes or alleles and tubal permeability were analyzed by the Pearson chi-square test with or without Yates correction. Results: There was no difference in CT antibody detection between the two groups. Women with obstructed tubes had a higher prevalence of being positive for the heterogenous genotype AB (36%) versus (16%) (p < 0.01). Similarly, the distribution of the normal A allele and variant B allele were also significant different between the two groups (p < 0,01). Conclusion: The findings suggest that while exposure to CT was similar in both groups of women the presence of the variant MBL B allele increases the risk for development of tubal obstruction, subsequent to a CT or other infection. In the Brazilian women evaluated possession of this genetic polymorphism increased the likelihood that blocked fallopian tubes will be a consequence of a genital tract infection

Objetivos: Verificar a frequência de Chlamydia trachomatis e a sua associação com a expressão de p16/Ki-67 em mulheres com lesões intraepiteliais cervicais. Analisar a associação entre a positividade para Chlamydia trachomatis e variáveis demográficas selecionadas, antecedentes sexuais e obstétricos, resultados anormais citológicos e anatomopatológicos. Também verificar a associação entre a expressão da dupla coloração para p16/Ki-67 com os resultados citológicos e anatomopatológicos. Métodos: Estudo de corte transversal em 1481 mulheres de 18 a 64 anos, participantes de projeto de rastreamento para câncer cervical realizado em São Paulo. As citologias foram coletadas em meio líquido e no líquido residual foi pesquisada a presença do DNA da Chlamydia trachomatis. Nos resultados de citologia anormal foi feita a pesquisa da expressão das proteínas p16/Ki-67. A análise estatística foi realizada usando-se os testes qui-quadrado e da razão de verossimilhanças. Foram estimados os valores de odds ratios (OR) com os respectivos intervalos com 95% de confiança. Resultados: A frequência de detecção da Chlamydia trachomatis foi 15,6%. Não houve associação estatisticamente significativa entre a presença de Chlamydia trachomatis e a expressão de p16/Ki-67 [OR=1,35 (0,5-3,4)]. A idade e o número de parceiros sexuais apresentou associação significativa com a presença de Chlamydia trachomatis [OR= 2,01 (1,1-3,6) e 4,14 (1,7-10,3)]. Não foi encontrada associação significativa entre citologia alterada e a positividade para Chlamydia trachomatis [1,21 (0,46-3,2)]. Não foi observada associação significativa entre os resultados anatomopatológicos e a presença de Chlamydia trachomatis (p = 0,112). A expressão do p16/Ki-67 mostrou associação estatisticamente significativa com lesões intraepiteliais cervicais de alto grau tanto nos resultados citológicos quanto anatomopatológicos. Conclusões: A frequência de infecção por Chlamydia trachomatis na amostra estudada foi de 15,6%. A associação da Chlamydia trachomatis com a dupla coloração para p16/Ki-67 nas citologias anormais não foi significativa, não sendo possível estabelecer uma associação clara entre a presença de Chlamydia trachomatis e a persistência da infecção por HPV oncogênico detectada por este marcador. Dentre as variáveis demográficas pesquisadas, a faixa etária apresentou associação estatisticamente significativa com a presença do DNA da Chlamydia trachomatis; mulheres com idade entre 35 e 45 apresentaram a maior frequência da infecção; entretanto, mesmo as outras faixas etárias mostraram uma alta frequência da presença desse patógeno; foi observada maior frequência da infecção entre as mulheres com mais de 10 parceiros sexuais, quando comparadas com as com menor número de parceiros durante a vida e essa associação foi estatisticamente significativa; não foi demonstrada associação significativa entre os resultados anormais da citologia com a positividade para a presença do DNA da Chlamydia trachomatis. Não foi encontrada associação significativa entre os resultados anatomopatológicos dirigidas pela colposcopia com a positividade da infecção pela bactéria. A positividade da dupla coloração para p16/Ki-67 foi significativamente maior nas lesões intraepiteliais cervicais de alto grau. Foi demonstrada associação estatisticamente significativa entre a expressão do p16/Ki-67 com os resultados anatomopatológicos das biopsias dirigidas pela colposcopia / Objective: To verify the frequency of Chlamydia trachomatis and its association with the expression of p16/Ki-67 on women with cervical intraepithelial lesions. To analyze the association between Chlamydia trachomatis presence and the selected demographic variables such as sexual and obstetric history, abnormal cytology and histopathology, as well as analyzing any association between the expression of dual staining for p16/Ki-67 with cytological and histopathological results. Methods: Cross-sectional study on 1481 women with ages between 18 and 64 years, who were enrolled in a screening project for cervical cancer held in São Paulo. The cytology was collected in liquid based medium and the residual liquid was submitted for examination to find the presence of Chlamydia trachomatis DNA. The expression of protein p16/Ki-67 was performed in the abnormal cytology results. Statistical analysis was performed using chi-square tests and likelihood ratio. The values of the odds ratios (OR) with respective intervals of 95% confidence were estimated. Results: The frequency of detection of Chlamydia trachomatis was 15.6%. There was no statistical significant association between the presence of Chlamydia trachomatis and the expression of p16/Ki-67 [OR = 1.35 (0.5 to 3.4)]. Both the age and number of sexual partners presented a significant association in presence of Chlamydia trachomatis [OR = 2.01 (1.1 to 3.6) and 4.14 (1.7 to 10.3)]. There was no significant association between abnormal cytology and positivity for Chlamydia trachomatis [1.21 (0.46 to 3.2)]. No significant association was found between histopathological results and presence of Chlamydia trachomatis (p= 0.112). The expression of p16/Ki-67 showed a significant statistical association with high grade intraepithelial lesions in both cytological and histopathological results. Conclusions: The prevalence of Chlamydia trachomatis infection on the sample studied was 15.6%. The association of Chlamydia trachomatis with dual staining for p16/Ki-67 in abnormal cytology was not significant, therefore, it is not possible to establish a clear association between the presence of Chlamydia trachomatis and the persistence of oncogenic HPV infection detected by this marker. Among the demographic variables, the age range showed statistically significant association with the presence of Chlamydia trachomatis; women aged between 35 - 45 years showed the highest rate of infection, nevertheless the other age ranges showed a high frequency of the presence of this pathogen; It has been observed a higher number of infected women who had more than 10 sexual partners compared to woman who had less partners throughout life and this association was statistically significant; No significant association was found between abnormal cytology with positivity for the presence of Chlamydia trachomatis DNA; There was no significant association between pathological results directed by colposcopy with the positivity of Chlamydia trachomatis; The positivity of the double staining for p16/Ki-67 was significantly higher in the cervical high-grade intraepithelial lesions; Statistical significant association was demonstrated between the expression of p16/Ki-67 with histopathological results of biopsy directed by colposcopy

Wie auch andere bakterielle Pathogene überträgt C. trachomatis Effektorproteine in die Wirtszelle, um deren Funktionen zu manipulieren. Das während der Invasion sekretierte Effektorprotein Tarp besitzt N-terminale SH2-Bindungsstellen und eine C-terminale SH3-Bindungsstelle für die Interaktion mit Wirtszellproteinen. Zur Bestimmung dieser Interaktionen wurden Protein-Microarrays mit nahezu alle humanen SH2- und SH3-Domänen verwendet. Zahlreiche neue Interaktionen wurden detektiert, wobei das Adaptorprotein SHC1 eine der stärksten SH2-abhängigen Interaktionen mit Tarp zeigte. Mittels Transkriptionsanalyse SHC1-abhängiger Genregulation während der Infektion konnten Gene identifiziert werden, welche an der Apoptose- und Zellwachstumskontrolle beteiligt sind. Infizierte Wirtszellen mit SHC1-Knockdown wiesen eine erhöhte Apoptoserate nach Stimulation mit TNF-alpha auf. Diese Ergebnisse offenbaren eine wichtige Rolle von SHC1 im Kontext des frühen, Chlamydien-induzierten Wirtszellüberlebens und deuten darauf hin, dass Tarp als vielseitige Signaltransduktionsplattform dient. Um Wirtszelllipide aufzunehmen, nutzt C. trachomatis Transportrouten der Wirtszelle und modifiziert Lipide bei der Aufnahme. Zur Bestimmung der Lipidzusammensetzung der Wirtszelle wurde diese mittels MALDI-TOF-Massenspektrometrie analysiert. Dabei hatte die Infektion den stärksten Einfluss auf Phosphatidylinoslitol (PI)- und Cardiolipin (CL)-Spezies. Des Weiteren konnten im Infektionsverlauf PI- und CL-Spezies mit einem Massenunterschied von 14 Da detektiert werden, was auf verzweigtkettige Fettsäurereste chlamydialen Ursprungs und eine Beteiligung der cytosolischen Phospholipase A2 (cPLA2) hindeutet. Entsprechend zeigten infizierte Zellen mit einem Knockdown von cPLA2 oder der Cardiolipinsynthase 1 eine signifikant reduzierte Bildung infektiöser Bakterien. Dies unterstreicht die Bedeutung von CL und einer funktionellen Nährstoffversorgung für die erfolgreiche Vermehrung von C. trachomatis. / Like many bacterial pathogens, C. trachomatis translocates effector proteins into the host cell to manipulate host cell functions. The early phase effector protein Tarp harbors N-terminal SH2 binding sites and a C-terminal SH3 binding site for the interaction with host cell proteins. To assess these interactions, protein microarrays comprising virtually all human SH2 and SH3 domains were used. Numerous novel interactions were discovered, while the adaptor protein SHC1 was among Tarp’s strongest SH2-dependent interaction partners. Transcriptome analysis of SHC1-dependent gene regulation during infection indicated that SHC1 regulates apoptosis- and growth-related genes. SHC1 knockdown sensitized infected host cells to TNF-alpha-induced apoptosis. These findings reveal a critical role for SHC1 in early Chlamydia-induced cell survival and suggest that Tarp functions as an important multivalent signaling hub. To acquire host-derived lipids, C. trachomatis hijacks cellular trafficking pathways and modifies lipids during the acquisition. To assess infection-dependent changes of the host cell lipid composition, cells were analyzed by MALDI-TOF mass spectrometry. Phosphatidylinositol (PI) and cardiolipin (CL) levels were most prominently influenced by C. trachomatis infection. Furthermore, PI and CL species with a 14 Da mass difference were detected during the course of infection, indicating the presence of Chlamydia-derived branched chain fatty acids and a role of cytosolic phospholipase A2 (cPLA2) in this process. Accordingly, infection of cPLA2 or cardiolipin synthase 1 knockdown cells resulted in a significantly reduced formation of infectious particles. These data demonstrate the importance of cardiolipin and a functional nutrient supply for the successful propagation of C. trachomatis.