Submitted by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2017-12-18T20:46:06Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Reprodução Não Autorizada.pdf: 47716 bytes, checksum: 0353d988c60b584cfc9978721c498a11 (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2017-12-18T20:46:43Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Reprodução Não Autorizada.pdf: 47716 bytes, checksum: 0353d988c60b584cfc9978721c498a11 (MD5) / Made available in DSpace on 2017-12-18T20:46:44Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Reprodução Não Autorizada.pdf: 47716 bytes, checksum: 0353d988c60b584cfc9978721c498a11 (MD5) Previous issue date: 2017-06-12 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Women in the gestation period undergo several physiological, immunological, hormonal and anatomical changes in their body, changes that leave them vulnerable to some infections. One of the pathogens that frequently infects women and which may have a considerable impact on pregnant women is the bacterium Chlamydia trachomatis (CT), whose infection is generally silent and can have serious consequences for the mother and fetus, such as premature birth, tubal pregnancy, and spontaneous abortion. The routine screening of this bacterium in women of childbearing age and in pregnant women has been carried out in some developed countries, but not yet in Brazil. The objective of this study was to study CT infection in pregnant women attended at the Basic Health Units of the Municipality of Coari, Amazonas, Brazil, and to verify possible associations between the presence of CT and clinical-epidemiological variables. It is a descriptive and cross-sectional study, with pregnant women attended at these units during the prenatal visit between July 2016 and March 2017. The pregnant women answered a questionnaire containing socio-demographic, behavioral and clinical data. The diagnosis of CT was performed using the polymerase chain reaction (PCR) technique in urine and cervix-vaginal samples. Of a total of 164 pregnant women selected, 100 were included in the study. The mean age was 22.87 years (SD = 6.24). The prevalence rate of CT infection was 18% (18/100). The prevalence of CT in the urine samples was 15% (95% CI: 8.65 - 23.53) and in the cervix-vaginal samples was 11% (95% CI: 5.62 - 18.83). The prevalence of CT was found in pregnant women with a mean age of 21.15 years (SD = 4.65), and was higher in those who started sexual activity before 15 years of age, and in the primigravida, but it was only Associated with those who had more than two partners in the last 12 months (p = 0.022) and those with gynecological complaint of "pain after intercourse" (p = 0.05). This study showed a high prevalence (18%) of CT infection among pregnant women in the municipality of Coari/AM, and reinforces the need to screen for this infection during prenatal care in this population. / As mulheres, no período da gestação, passam por diversas alterações fisiológicas, imunológicas, hormonais e anatômicas em seu corpo, alterações estas que as deixam vulneráveis a algumas infecções. Um dos patógenos que frequentemente infecta mulheres e que pode ter considerável impacto sobre gestantes é a bactéria Chlamydia trachomatis (CT), cuja infecção é geralmente silenciosa e pode trazer sérias consequências para a mãe e o feto, tais como o parto prematuro, gravidez tubária e aborto espontâneo. O rastreamento rotineiro dessa bactéria em mulheres em idade fértil e em gestantes tem sido realizado em alguns países desenvolvidos, mas ainda não no Brasil. O objetivo desta pesquisa foi estudar a infecção por CT em gestantes atendidas nas Unidades Básicas de Saúde do Município de Coari, Amazonas, Brasil, e verificar possíveis associações entre a presença de CT e variáveis clínico-epidemiológicas. Trata-se de um estudo descritivo e transversal, com gestantes atendidas nessas unidades durante a consulta de pré-natal, entre julho de 2016 e março de 2017. As gestantes responderam um questionário contendo dados sócio-demográficos, comportamentais e clínicos. O diagnóstico da CT foi realizado pela técnica de reação em cadeia da polimerase (PCR) em amostras de urina e cérvico-vaginais. De um total de 164 gestantes selecionadas, 100 foram incluídas no estudo. A média de idade foi de 22,87 anos (DP= 6,24). A taxa de prevalência da infecção por CT encontrada foi de 18% (18/100). A prevalência da CT nas amostras de urina foi de 15% (IC95%: 8,65 – 23,53) e nas amostras cérvico-vaginais foi de 11% (IC95%: 5,62 – 18,83). A prevalência da CT foi encontrada nas gestantes com média de idade de 21,15 anos (DP=4,65), e foi maior nas que tiveram o início da atividade sexual antes dos 15 anos de idade, e nas primigesta, no entanto, somente associada com as que tiveram mais de dois parceiros nos últimos 12 meses (p= 0,022) e as que apresentaram queixa ginecológica “dor após a relação sexual” (p= 0,05). Este estudo mostrou uma alta prevalência (18%) da infecção causada por CT entre gestantes do município de Coari/AM, e reforça a necessidade do rastreio dessa infecção durante o pré-natal nessa população.

Introduction Yearly, 90 million people are infected with C. trachomatis. Even though it is easily treated with antibiotics the often-asymptomatic infection often spreads prior to detection. A vaccine is therefore of great interest. A chimeric major outer membrane protein (MOMP) of C. trachomatis has in earlier studies proved to contain the epitopes necessary for immunization. In this thesis the chimeric MOMP gene was cloned and expressed in E. coli. Furthermore, the expression of the protein was analyzed in previously transformed A. thaliana. Materials and Methods The chimeric MOMP gene was cloned into E.coli. Following vector amplification, the gene was expressed and the protein purified by affinity chromatography.  Seeds from different lines of previously transformed A. thaliana were screened by PCR. Hits were then analyzed by western blot.  Results The results show successful cloning and expression of the chimeric MOMP gene in E. coli. The following protein purification did result in purified protein, however in low concentration. For the A.thaliana lines, the presence and correct orientation of the gene was verified in some of the lines screened. The B7 line was verified to express the protein. Discussion The low concentration of purified protein in E.coli was probably due to un-optimized imunnoprecipitation conditions. In expression analysis of A. thaliana, purification of plant samples by immunoprecipitation prior to running western blot gave results, whereas running un-purified samples in urea buffer did not, probably due to interfering proteins in wild type plants.

Chlamydia trachomatis est une bactérie intracellulaire obligatoire pathogène pour l'homme, qui se développe dans un compartiment appelé inclusion. La membrane de l'inclusion constitue une protection contre les défenses de l'hôte, mais limite l'accès aux nutriments. Un élément essentiel pour C. trachomatis est le glucose. Son polymère, le glycogène, est abondant dans le lumen de l'inclusion. Ce travail a eu pour objectif de reconstituer le flux de glucose dans des cellules infectées et d'expliquer l'accumulation du glycogène. En résumé, notre travail démontre que l'accumulation de glycogène dans la lumière de l'inclusion est le résultat de deux processus, l'import de glycogène " brut " de l'hôte par invagination de la membrane de l'inclusion, et la synthèse de novo de glycogène dans le lumen de l'inclusion. Ce dernier implique l'import d'UDP-glucose par un transporteur de la cellule hôte qui est recruté dans la membrane de l'inclusion, et la sécrétion d'enzymes bactériennes dans le lumen de l'inclusion. Ces mécanismes permettent aux bactéries de stocker des molécules énergétique, inaccessibles à l'hôte. / The human pathogen Chlamydia trachomatis is an obligate intracellular bacterium, which develops in a parasitophorous compartment called inclusion. The inclusion membrane serves as a barrier to host defense mechanisms, but limits access to nutrients. One essential nutrient for C. trachomatis is glucose, and its polymer, glycogen, is highly abundant in the inclusion lumen. This work aimed to reconstitute the glucose flow in C. trachomatis infected cells and to understand the mechanisms for glycogen accumulation. In summary, our work demonstrates that glycogen storage in C. trachomatis inclusions is the result of two different strategies, bulk acquisition of host glycogen through invagination of the inclusion membrane, and de novo synthesis of glycogen within the inclusion lumen. The latter mechanism implicates the import of host UDP-glucose through a host transporter that is recruited to the inclusion membrane, and the secretion of bacterial glycogen enzymes into the inclusion lumen. These processes allow the bacteria to build an energy store within the inclusion lumen, out of reach for the host.

Chlamydia trachomatis, the most common bacterial sexually transmitted disease agent worldwide, enters a viable, non-dividing and non-infectious state (historically termed persistence and more recently referred to as the chlamydial stress response) when exposed to penicillin G in culture. Notably, penicillin G-exposed chlamydiae can reenter the normal developmental cycle upon drug removal and are resistant to azithromycin-mediated killing. Because penicillin G is less frequently prescribed than other ß-lactams, the clinical relevance of penicillin G-induced chlamydial persistence/stress has been questioned. The goal of this study was to determine whether more commonly used penicillins also induce C. trachomatis serovar E persistence/stress. All penicillins tested, as well as clavulanic acid, induced formation of aberrant, enlarged reticulate bodies (RB) (called aberrant bodies or AB) characteristic of persistent/stressed chlamydiae. Exposure to the penicillins and clavulanic acid also reduced chlamydial infectivity by >95%. None of the drugs tested significantly reduced chlamydial unprocessed 16S rRNA or genomic DNA accumulation, indicating that the organisms were viable, though non-infectious. Finally, recovery assays demonstrated that chlamydiae rendered essentially non-infectious by exposure to ampicillin, amoxicillin, carbenicillin, piperacillin, penicillin V, and clavulanic acid recovered infectivity after antibiotic removal. These data definitively demonstrate that several commonly used penicillins induce C. trachomatis persistence/stress at clinically relevant concentrations.

The Chlamydiaceae are widespread pathogens of both humans and animals. Chlamydia trachomatis infection causes blinding trachoma and reproductive complications in humans. Chlamydia pneumoniae causes human respiratory tract infections and atypical pneumonia. Chlamydia suis infection is associated with conjunctivitis, diarrhea, and failure to gain weight in domestic swine. Chlamydial infections in humans and domesticated animals are generally controlled by antibiotic treatment—particularly macrolides (usually azithromycin) and tetracyclines (tetracycline and doxycycline). Tetracycline-containing feed has also been used to limit infections and promote growth in livestock populations, although its use has decreased because of growing concerns about antimicrobial resistance development. Because Sandoz and Rockey published an elegant review of chlamydial anti-microbial resistance in 2010, we will review the following: (i) antibiotic resistance in C. suis, (ii) recent evidence for acquired resistance in human chlamydial infections, and (iii) recent non-genetic mechanisms of antibiotic resistance that may contribute to treatment failure.

Confinement of the obligate intracellular bacterium Chlamydia trachomatis to a membrane-bound vacuole, termed an inclusion, within infected epithelial cells neither prevents secretion of chlamydial antigens into the host cytosol nor protects chlamydiae from innate immune detection. However, the details leading to chlamydial antigen presentation are not clear. By immunoelectron microscopy of infected endometrial epithelial cells and in isolated cell secretory compartments, chlamydial major outer membrane protein (MOMP), lipopolysaccharide (LPS) and the inclusion membrane protein A (IncA) were localized to the endoplasmic reticulum (ER) and co-localized with multiple ER markers, but not with markers of the endosomes, lysosomes, Golgi nor mitochondria. Chlamydial LPS was also co-localized with CD1d in the ER. Since the chlamydial antigens, contained in everted inclusion membrane vesicles, were found within the host cell ER, these data raise additional implications for antigen processing by infected uterine epithelial cells for classical and non-classical T cell antigen presentation.

A common model for studying Chlamydia trachomatis and growing chlamydial stocks uses Lymphogranuloma venereum serovar L2 and non-polarized HeLa cells. However, recent publications indicate that the growth rate and progeny yields can vary considerably for a particular strain depending on the cell line/type used, and seem to be partially related to cell tropism. In the present study, the growth of invasive serovar L2 was compared in endometrial HEC-1B and endocervical HeLa cells polarized on collagen-coated microcarrier beads, as well as in HeLa cells grown in tissue culture flasks. Microscopy analysis revealed no difference in chlamydial attachment/entry patterns or in inclusion development throughout the developmental cycle between cell lines. Very comparable growth curves in both cell lines were also found using real-time PCR analysis, with increases in chlamydial DNA content of 400-500-fold between 2 and 36 h post-inoculation. Similar progeny yields with comparable infectivity were recovered from HEC-1B and HeLa cell bead cultures, and no difference in chlamydial growth was found in polarized vs. non-polarized HeLa cells. In conclusion, unlike other C. trachomatis strains such as urogenital serovar E, invasive serovar L2 grows equally well in physiologically different endometrial and endocervical environments, regardless of the host cell polarization state.

Several chlamydial antigens have been detected in the infected epithelial cell cytosol and on the host cell surface prior to their presumed natural release at the end of the 72-96 h developmental cycle. These extra-inclusion antigens are proposed to influence vital host cell functions, antigen trafficking and presentation and, ultimately, contribute to a prolonged inflammatory response. To begin to dissect the mechanisms for escape of these antigens from the chlamydial inclusion, which are enhanced on exposure to antibiotics, polarized endometrial epithelial cells (HEC-1B) were infected with Chlamydia trachomatis serovar E for 36 h or 48 h. Infected cells were then exposed to chemotactic human polymorphonuclear neutrophils not loaded or pre-loaded in vitro with the antibiotic azithromycin. Viewed by electron microscopy, the azithromycin-mediated killing of chlamydiae involved an increase in chlamydial outer membrane blebbing followed by the appearance of the blebs in larger vesicles (i) everting from but still associated with the inclusion as well as (ii) external to the inclusion. Evidence that the vesicles originated from the chlamydial inclusion membrane was shown by immuno-localization of inclusion membrane proteins A, F, and G on the vesicular membranes. Chlamydial heat shock protein 60 (chsp60) copies 2 and 3, but not copy 1, were released from RB and incorporated into the everted inclusion membrane vesicles and delivered to the infected cell surface. These data represent direct evidence for one mechanism of early antigen delivery, albeit membrane-bound, beyond the confines of the chlamydial inclusion.

Chlamydial attachment and infectivity in vitro and ascending disease and sequelae in vivo have been reported to be enhanced/modulated by estrogen. Endometrial carcinoma cell lines Ishikawa and HEC-1B and the breast cancer lines MCF-7 and HCC-1806 were examined for Chlamydia trachomatis E infectivity. Estrogen receptor (ER) presence was confirmed by Western blot and qRT-PCR analyses. FACS analysis was used to determine the percent of plasma membrane-localized ERs (mERs), and their activity was tested by estrogen binding and competitive estrogen antagonists assays. Chlamydiae grew in all cell lines with HEC (90%) ≫ MCF-7 (57%) > Ishikawa (51%) ≫ HCC-1806 (20%). The cell line ER isoform composition was re-defined as: ERα + ERβ + for MCF-7, HCC-1806 and Ishikawa; and ERβ only for HEC-1B. HeLa cells were also tested and found to express ERβ, but not ERα. A small percentage of both ERs were surface-exposed and functionally active. The endometrium- predominant ERβ isoform was found in all cell lines, including those most representative of the common sites of C. trachomatis infection. Thus, the role of chlamydial attachment/infectivity will now be analyzed in ERβ + and - isogenic HEC-1B cells.

Recent Findings: Recent data support previous work indicating that estrogen enhances chlamydial development via multiple mechanisms. Progesterone negatively impacts Chlamydia infections also through multiple mechanisms, particularly by altering the immune response. Conflicting data exist regarding the effect of synthetic hormones, such as those found in hormonal contraceptives, on chlamydial infections. Summary: Numerous studies over the years have indicated that female sex hormones affect C. trachomatis infection. However, we still do not have a clear understanding of how these hormones alter Chlamydia disease transmission and progression. The studies reviewed here indicate that there are many variables that determine the outcome of Chlamydia/hormone interactions, including (1) the specific hormone, (2) hormone concentration, (3) cell type or area of the genital tract, (4) hormone responsiveness of cell lines, and (5) animal models.