Neisseria gonorrhoeae verursacht die sexuell übertragbare Krankheit Gonorrhoe und ist ein Typ-IV-Pili (Tfp) exprimierendes Bakterium, das den Urogenitaltrakt besiedelt. Frühe Infektionsstadien piliierter N. gonorrhoeae (P+GC) sind durch die Tfp-vermittelte Adhärenz an Wirtszellen gekennzeichnet, dann erfolgt die Bildung von Mikrokolonien auf Wirtszellepithelien. Hier wird gezeigt, dass die Wirtszellen an der effizienten Bildung der extrazellulären Mikrokolonien beteiligt sind. P+GC die fixierte Wirtszellen infizieren weisen eine verzögerte Mikrokoloniebildung gegenüber einer Infektion lebender Wirtszellen auf. Kortikales Aktin wird zusammen mit Signalproteinen innerhalb der Wirtszellen zu den adhärierten Bakterien rekrutiert, darunter das Hauptstrukturprotein von Caveolae-Membrandomänen, Caveolin-1 (Cav1). Eine Reduzierung der Expression von Cav1 führt zu einer verstärkten Aufnahme von P+GC in die Wirtszellen, wohingegen die Expression von Cav1 in Cav1-negativen Zellen eine Internalisierung verhindert. Internalisierte Bakterien weisen dabei geringere Überlebensraten auf je länger sie in den Wirtszellen verbleiben. Die Rekrutierung von Cav1 ist eine unmittelbare und kontinuierliche zelluläre Antwort auf eine Infektion mit P+GC, welche die Phosphorylierung von Cav1 an Tyrosin 14 bedingt. Zusätzlich erforderte die Cav1-vermittelte Blockierung der Internalisierung der Bakterien und die Verankerung von Cav1 mit dem Zytoskelett eine Tyrosinphosphorylierung von Cav1. Eine Analyse möglicher Interaktionspartner von phosphoryliertem Cav1 zeigte eine direkte Interaktion mit Vav2. Sowohl Vav2 als auch sein Substrat, die kleine GTPase RhoA, blockieren die Aufnahme von Bakterien in die in Wirtszellen. Die Aktivierung von RhoA nach P+GC Infektion erfordert die Expression von Cav1, was auf einen Cav1-Vav2-RhoA Signalweg hindeutet. Darüber hinaus wurden in dieser Arbeit sechs neue, eine SH2-Domäne-beinhaltende Interaktionspartner von phosphoryliertem Cav1 identifiziert. / Neisseria gonorrhoeae causes the sexually transmitted disease gonorrhea and colonizes mucosal epithelia of the human urogenital tract. The early stages of infection with piliated N. gonorrhoeae (P+GC) are characterized by Tfp-mediated adherence to host cells, followed by formation of bacterial microcolonies on the surface of host cells. This study provides evidence that host cell participation is required for the efficient formation of extracellular microcolonies during Neisseria infection. P+GC infecting fixed host cells demonstrate altered motility and delayed microcolony formation compared to infecting living host cells. Cortical actin and various signal transducing proteins are recruited to the site of bacterial attachment within host cells, one of them being the major structural protein of plasma membrane caveolae, Caveolin-1 (Cav1). Down-regulation of Cav1 results in increased uptake of P+GC into host cells whereas expression of the protein in Cav1-negative cells blocks bacterial internalization. Host cell entry results in decreased viability of internalized bacteria over time. Cav1 recruitment is demonstrated to be an immediate and continuous cellular response to P+GC infection that involves Cav1 phosphorylation on its tyrosine 14 residue. Prevention of bacterial uptake mediated by Cav1 as well as tight association of Cav1 with the cytoskeleton also requires tyrosine phosphorylation. A broad analysis of interaction partners of phosphorylated Cav1 revealed a direct interaction with the Rho-family guanine nucleotide exchange factor Vav2. Both Vav2 and its substrate, the small GTPase RhoA, are involved in preventing bacterial uptake and RhoA activation after P+GC infection requires Cav1 expression, thus providing evidence for a Cav1-Vav2-RhoA signaling cascade. Moreover, six novel SH2-domain containing interaction partners of tyrosine phosphorylated Cav1 have been identified, all of which have been implicated in modulating the cytoskeleton.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2007. / Härtill 4 uppsatser.

Made available in DSpace on 2016-12-23T13:56:05Z (GMT). No. of bitstreams: 1 Marcelo Joaquim Barbosa.pdf: 493310 bytes, checksum: 1cc616c331737b5497291791ef93fb83 (MD5) Previous issue date: 2010-07-12 / Objetivos: Determinar a prevalência de Neisseria gonorrhoeae (NG) e Chlamydia trachomatis (CT) e identificar fatores demográficos, comportamentais e clínicos correlacionados a essas infecções em homens atendidos em seis clínicas de DST no Brasil. Métodos: Estudo multicêntrico, em corte transversal realizado em homens que procuraram atendimento em clínicas de DST. O estudo incluiu clínicas de DST em seis cidades distribuídas nas cinco macrorregiões do Brasil, 2004/2005. Metodologia: Coletou-se 20 ml do primeiro jato de urina para testar NG e CT por DNA-PCR. Resultados: Um total de 767 (92,9%) homens foi incluído no estudo. A mediana de idade foi de 24 (DIQ21 30) anos. A prevalência de infecção por CT foi 13,1% (IC95% 10,7%-15,5%) e por NG 18,4% (IC95% 15,7%-21,1%). A prevalência de coinfecção foi 4,4% (IC95% 2,95%-5,85%). Os fatores identificados como sendo independentemente associados com a infecção por clamídia no modelo final de regressão logística foram: ser jovem (15-24 anos) [OR=1,4 (IC95% 1,01-1,91)], apresentar corrimento uretral ao exame [OR=4.8 (IC95% 1,52-15,05)], verrugas genitais [OR=3,0 (IC95% 1,49-5,92)] e história prévia de corrimento uretral [OR=2,4 (IC95% 1,11-5,18)]. As variáveis associadas com gonorréia foram: ser jovem (15-24 anos) [OR=1,5 (IC95% 1,09-2,05)], apresentar corrimento uretral ao exame [OR=9,9 (IC95% 5,53-17,79)], verrugas genitais [OR=18,3 (IC95% 8,03-41,60)] e úlcera ao exame clínico [OR=4,9 (IC95% 1,06-22,73)]. Conclusões: Estes resultados mostram implicações importantes na realização de diagnóstico e tratamento precoces para evitar a transmissão, complicações e implementação de medidas de educação em saúde direcionadas aos homens e deve ser estimulado o oferecimento de testes para DST na rotina dos nos serviços. / Objectives: To determine the prevalence of Neisseria gonorrhoeae(NG) and Chlamydia trachomatis (CT) infections and identified demographic, behavioral, and clinical correlates of these infections in men attending six STI clinics in Brazil. Methods: Multicentric, cross-sectional study performed among men attending STI clinics in Brazil. The study included STD clinics in six diverse cities in the five geographic regions of Brazil in 2005. Patients provided 20 ml of first catch urine for testing for NG and CT by DNA-PCR. Results: A total of 767 (92.9%) men were included in the study. The median age was 24 (IQR 21 30) years old. Prevalence of Chlamydia infection was 13.1% (95%CI 10.7%-15.5%) and gonorrhea was 18.4% (95%CI 15.7%-21.1%). Coinfection prevalence was 4.4% (95%CI 2.95%-5.85%) in men who sought assistance in STI clinics. Factors identified as being independently associated with Chlamydia trachomatis in the final multiple logistic model were being younger (15-24) [OR=1.4 (95%CI 1.01-1.91)], present urethral discharge [OR=4.8 (95%CI 1.52-15.05)], genital warts [OR=3.0 (95%CI 1.49-5.92)] and previous history of urethral discharge [OR=2.4 (95%CI 1.11-5.18)]. Variables associated with gonorrhea were being younger (15 to 24) [OR=1.5 (95%CI 1.09-2.05)], presence of urethral discharge [OR=9.9 (95%CI 5.53-17.79)], genital warts [OR=18.3 (95%CI 8.03-41.60)] and ulcer in clinical examination [OR=4.9 (95%CI 1.06-22.73)]. Conclusions: These findings have important implications for implementing education and prevention efforts directed toward men at risk of HIV/STD. A venue-based approach to offer routinely testing to men in STD clinic should be stimulated.

Made available in DSpace on 2016-12-23T13:56:06Z (GMT). No. of bitstreams: 1 Dissertacao de Marcelo Joaquim Barbosa.pdf: 493316 bytes, checksum: 05c09f00c2dba0603712d1b6f468b750 (MD5) Previous issue date: 2010-07-12 / Objetivos: Determinar a prevalência de Neisseria gonorrhoeae (NG) e Chlamydia trachomatis (CT) e identificar fatores demográficos, comportamentais e clínicos correlacionados a essas infecções em homens atendidos em seis clínicas de DST no Brasil. Métodos: Estudo multicêntrico, em corte transversal realizado em homens que procuraram atendimento em clínicas de DST. O estudo incluiu clínicas de DST em seis cidades distribuídas nas cinco macrorregiões do Brasil, 2004/2005. Metodologia: Coletou-se 20 ml do primeiro jato de urina para testar NG e CT por DNA-PCR. Resultados: Um total de 767 (92,9%) homens foi incluído no estudo. A mediana de idade foi de 24 (DIQ21 30) anos. A prevalência de infecção por CT foi 13,1% (IC95% 10,7%-15,5%) e por G 18,4% (IC95% 15,7%-21,1%). A prevalência de coinfecção foi 4,4% (IC95% 2,95%-5,85%). Os fatores identificados como sendo independentemente associados com a infecção por clamídia no modelo final de regressão logística foram: ser jovem (15-24 anos) [OR=1,4 (IC95% 1,01-1,91)], apresentar corrimento uretral ao exame [OR=4.8 (IC95% 1,52-15,05)], verrugas genitais [OR=3,0 (IC95% 1,49-5,92)] e história prévia de corrimento uretral [OR=2,4 (IC95% 1,11-5,18)]. As variáveis associadas com gonorréia foram: ser jovem (15-24 anos) [OR=1,5 (IC95% 1,09-2,05)], apresentar corrimento uretral ao exame [OR=9,9 (IC95% 5,53-17,79)], verrugas genitais [OR=18,3 (IC95% 8,03-41,60)] e úlcera ao exame clínico [OR=4,9 (IC95% 1,06-22,73)]. Conclusões: Estes resultados mostram implicações importantes na realização de diagnóstico e tratamento precoces para evitar a transmissão, complicações e implementação de medidas de educação em saúde direcionadas aos homens e deve ser estimulado o oferecimento de testes para DST na rotina dos nos serviços / Objectives: To determine the prevalence of Neisseria gonorrhoeae(NG) and Chlamydia trachomatis (CT) infections and identified demographic, behavioral, and clinical correlates of these infections in men attending six STI clinics in Brazil. Methods: Multicentric, cross-sectional study performed among men attending STI clinics in Brazil. The study included STD clinics in six diverse cities in the five geographic regions of Brazil in 2005. Patients provided 20 ml of first catch urine for testing for NG and CT by DNA-PCR. Results: A total of 767 (92.9%) men were included in the study. The median age was 24 (IQR 21 30) years old. Prevalence of Chlamydia infection was 13.1% (95%CI 10.7%-15.5%) and gonorrhea was 18.4% (95%CI 15.7%-21.1%). Coinfection prevalence was 4.4% (95%CI 2.95%-5.85%) in men who sought assistance in STI clinics. Factors identified as being independently associated with Chlamydia trachomatis in the final multiple logistic model were being younger (15-24) [OR=1.4 (95%CI 1.01-1.91)], present urethral discharge [OR=4.8 (95%CI 1.52-15.05)], genital warts [OR=3.0 (95%CI 1.49-5.92)] and previous history of urethral discharge [OR=2.4 (95%CI 1.11-5.18)]. Variables associated with gonorrhea were being younger (15 to 24) [OR=1.5 (95%CI 1.09-2.05)], presence of urethral discharge [OR=9.9 (95%CI 5.53-17.79)], genital warts [OR= 8.3 (95%CI 8.03-41.60)] and ulcer in clinical examination [OR=4.9 (95%CI 1.06-22.73)]. Conclusions: These findings have important implications for implementing education and prevention efforts directed toward men at risk of HIV/STD. A venue-based approach to offer routinely testing to men in STD clinic should be stimulated

Orientadores: Paulo César Giraldo, Ana Katherine da Silveira Gonçalves / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-16T20:57:25Z (GMT). No. of bitstreams: 1 Amaral_RoseLuceGomesdo_D.pdf: 1220448 bytes, checksum: d6f8f0907e1226cc41668603ab978683 (MD5) Previous issue date: 2010 / Resumo: O uso de duchas vaginais é comum em diferentes povos e culturas. Entre as mulheres que a praticam, a maioria a faz após a menstruação, antes ou após a relação sexual, com a finalidade de evitar o odor desagradável ou para eliminar corrimentos, refrescar a genitália ou prevenir a gravidez. Não está claro na literatura se as duchas poderiam causar problemas às usuárias. Objetivo: Verificar se o uso habitual de duchas vaginais associa-se à infecção genital por Chlamydia trachomatis/ Neisseria gonorrhoeae e Papilomavírus humano (HPV) em mulheres profissionais do sexo. Sujeitos e Métodos: Estudo de corte transversal avaliou 200 mulheres - 111 profissionais do sexo (PS) e 89 não profissionais do sexo (NPS) - assistidas em uma Unidade Básica de Saúde de Campinas, São Paulo, Brasil. Todas as mulheres foram entrevistadas e examinadas por um único pesquisador. A anamnese abordou os antecedentes demográficos, higiênicos, sexuais e médicos, como idade, cor, paridade, escolaridade, tabagismo, estado civil, uso de duchas vaginais, idade da primeira relação sexual, número de parceiros sexuais, número de coitos por semana, práticas sexuais, uso de preservativo e lubrificante. Amostras de células cervicais foram coletadas para testes de captura híbrida objetivando Chlamydia trachomatis/Neisseria gonorrhoaea e HPV. Na análise estatística usou-se o teste exato de Fisher ou X2 para as variáveis discretas, e Mann-Whitney para as variáveis não-paramétricas. O estudo foi aprovado pelo Comitê de Ética em Pesquisa (no 902/2009) e contou com a colaboração da Secretaria de Saúde do Estado de São Paulo e Laboratório Digene. Resultado: Cerca de 40% das mulheres investigadas praticavam duchas vaginais três ou mais vezes por semana (61,7% das PS e somente 14,6% das NPS). A infecção por CT/NG foi positiva em 10,5% do total dos casos, 17 casos (15,3%) em PS e em quatro casos (4,5%) das NPS (p=0,01), porém não houve diferenças significativas entre usuárias de duchas vaginais (14,81%) e não usuárias (7,6%), (p=ns). As PS não usuárias de duchas vaginais tiveram quase o dobro de infecção CT/NG que as NPS usuárias de duchas vaginais (13,9% vs 7,7%). O HPV foi positivo em 40,5% dos casos, sendo 55,8% da PS e em 21,3% das NPS (p=0,001), contudo DNA-HPV não foi significativamente diferente (p=0,47) entre PS usuárias de duchas vaginais (54,4%) e em não usuárias (58,1%). HPV de alto risco foi positivo em 16,2% e 11,6% (p=ns) e o HPV de baixo risco em 23,5% e 30,2% (p=ns) em usuárias e não usuárias de duchas vaginais respectivamente. Os HPV de alto e baixo riscos foram encontrados simultaneamente em 14,7% e 16,2% das usuárias e não usuárias de duchas vaginais, respectivamente (p=ns). Conclusão: O uso de duchas vaginais não se associou às infecções genitais por CT/NG e HPV de alto ou baixo grau nas mulheres estudadas (profissionais do sexo e não profissionais do sexo) / Abstract: The use of vaginal douching (VD) is widespread around the world, and is more common than is to be expected. The majority of women douche after menses, before or after sexual intercourse to prevent odor, to alleviate vaginal symptoms, or to prevent pregnancy, however it is not clear in the literature if this habit can cause damages for women. Objective: Establish whether high frequency VD favors Chlamydia trachomatis (CT)/Neisseria gonorrhoeae (NG) and Papilomavírus humano (HPV) infection in female sex workers (SW). Subjects and Methods: A clinical cross-sectional study involving 200 women, 111 SW and 89 non-sex workers (NSW) in a Health Center in Brazil. The subjects were submitted to an interview and examined by a single researcher. A questionnaire was filled out with data that included, medical and demographic history (age, race, parity, education, smoking, marital status), hygiene (use of VD, frequency and the solution used) and sexual behaviour (first sexual relation, number of sexual partners, frequency, sexual practices, homosexual relationships, use of condom and lubricant). Cervical samples were collected for CT and NG testing by hybrid capture 2 assay. Statistical analysis used the Fisher's exact test or qui square for discrete variables and Mann-Whitney test for nonparametric variables. The study was approved by Committee the Ethics in Research (no 902/2009) and received the cooperation of São Paulo Secretariat of Health and Digene laboratory. Results: Approximately 40% of women practiced douche three or more times per week (61.7% of SW and only 14.6% of the NSW). Infection with CT / NG was positive in 10.5% of the total cases, 17 cases (15.3%) in SW and in four cases (4.5%) of the NSW (p = 0.01). However, It was in 14.81% of D and but in and 7.6% of ND (p = ns). SW douchers had almost double CT and NG infections than NSW douchers (13.9% vs 7.7%). HPV infection was detected in 40.5% of cases, 55.8% of SW and 21.3% of NSW of (p = 0.001), despite of DNA HPV was not different (p = 0.47) between SW douchers (54.4%) and non-douchers (58.1%). High-risk DNA-HPV was positive in 16.2% and 11.6% (p = ns) and low-risk DNA-HPV in 23.5% and 30.2% (p = ns) in douchers and non-douchers respectively. High and low risk DNA-HPV were found simultaneously in 14.7% and 16.2% of douchers and non-douchers respectively (p = ns). Conclusion: The use of VD is not a causal factor for cervical CT/NG and High-/low risk HPV infection in women studied (SW and NSW) / Doutorado / Tocoginecologia / Doutor em Tocoginecologia

Made available in DSpace on 2014-07-29T15:30:37Z (GMT). No. of bitstreams: 1 Dissertacao Jannaina Karlla de Queiroz Duarte.pdf: 1435551 bytes, checksum: 9764e4445592cad24997ac8311dc77d7 (MD5) Previous issue date: 2008-08-29 / BACKGROUND: Adolescents are at great risk for genital Neisseria gonorrhoeae infection. Although most infections are asymptomatic it can cause severe reproductive sequels to women. Few studies exist about prevalence of these infection in Brazil and fewer in adolescent population. OBJECTIVES: To determine the prevalence of Neisseria gonorrhoeae genital infection among female sexually active adolescents, in Distrito Sanitário Noroeste from Goiânia, Brazil and to identify demographic and behavioral profiles associated to gonococcal infection. METHODS: We performed a cross-sectional study among 427 sexually active female adolescents (15-19 years), random selected at Distrito Sanitário Noroeste and served by the Health Family Program. It were excluded pregnant adolescents, pos-partum, using antibiotics or on period. The gonococcal infection was determined by using polimerase chain reaction (PCR) of Amplicor Roche kit for N. gonorrhoeae and C. trachomatis applied to endocervical swab specimens. Sociodemographic and behavioral data were assessed by face-to-face questionnaire. RESULTS: The mean age of 427 random selected adolescents was 17.2 ± 1.3 years, most female were single (67.9%). The mean age of menarch was 12.4 years and at first sexual intercourse was 15 years. 35,1% had the first sexual intercourse before 15 years and 16,2% reported more than 3 partners in life. About 80% of the participants reported inconsistent condom use. Among those with gonococcal infection, 22,2% also had chlamydial infection. The prevalence of gonococcal infection was 2,1% (95% CI 1,0 4,1). CONCLUSIONS: Adolescents studied presented risk sexual behaviors to sexually transmitted diseases. These results demonstrate the need for preventive medicine programs for adolescents females with ongoing STI prevention activities. / INTRODUÇÃO: Os adolescentes constituem o grupo de maior risco para a infecção genital por Neisseria gonorrhoeae. Embora a maioria das infecções sejam assintomáticas podem causar graves seqüelas reprodutivas nas mulheres. Existem poucos estudos sobre a prevalência desta infecção no Brasil e menos ainda neste grupo populacional. OBJETIVOS: Determinar a prevalência da infecção genital por N. gonorrhoeae em adolescentes sexualmente ativas residentes no Distrito Sanitário Noroeste do município de Goiânia e identificar o perfil sócio-demográfico e comportamental das adolescentes estudadas. METODOLOGIA: Foi realizado um estudo de corte transversal em 427 adolescentes do gênero feminino com idade entre 15 e 19 anos, sexualmente ativas, selecionadas aleatoriamente no Distrito Sanitário Noroeste e atendidas pelo Programa de Saúde da Família. Foram excluídas adolescentes grávidas ou no puerpério, em uso de antibióticos ou menstruadas. O diagnóstico da infecção gonocócica foi realizado empregando a Reação em Cadeia da Polimerase (PCR) em amostras endocervicais utilizando o kit Amplicor Roche para N. gonorrhoeae e C. trachomatis. Foram também obtidas informações sóciodemográficas e de comportamento sexual através de questionário na forma de entrevista. RESULTADOS: A média de idade das 427 adolescentes selecionadas foi de 17,2 ± 1,3 anos, sendo a maioria delas solteira (67,9%). A média de idade da primeira relação sexual foi de 15 anos. 35,1% das adolescentes tiveram a primeira relação sexual antes dos 15 anos e 16,2% relataram mais que três parceiros durante a vida. Cerca de 80% relataram uso inconsistente de preservativo. Dentre aquelas com infecção gonocócica, 22,2% tinham também infecção clamidial. A prevalência encontrada para a infecção gonocócica foi de 2,1% (IC 95% 1,0 4,1). CONCLUSÕES: As adolescentes estudadas apresentaram comportamentos sexuais de risco para a aquisição de DST. Estes dados mostram a necessidade de programas de medicina preventiva direcionados à adolescentes, principalmente atividades de prevenção de DST.

The obligate human pathogen Neisseria gonorrhoeae successfully overcomes host strategies to limit essential nutrients, termed “nutritional immunity” by expression of TonB-dependent transporters (TdTs): outer membrane receptors that facilitate nutrient transport in an energy-dependent manner. N. gonorrhoeae encodes eight TdTs, five of which facilitate utilization of iron or iron-chelates from host derived proteins including transferrin, lactoferrin and hemoglobin, in addition to siderophores from neighboring bacteria. The transferrin utilization system was previously shown to be critical for establishing infection in human males; demonstrating the possible contributions of TdTs to gonococcal pathogenesis. As such, studies describing the biological function and contribution to pathogenesis of the remaining three uncharacterized TdTs (TdfG, TdfH and TdfJ) are needed. In this study we report that neither TdfG, TdfH nor TdfJ are heme receptors as gonococcal heme utilization occurs passively, independent of energy derived from the TonB system. We also report that TdfH and TdfJ are zinc (Zn) regulated and identify virulence associated regulators that modulate expression of these TdTs, which is in some cases strain-specific. We report that both TdfH and TdfJ contribute to Zn acquisition in N. gonorrhoeae and we characterize TdfH as a calprotectin receptor. Calprotectin, an immune effector protein highly expressed in neutrophils, has antimicrobial activity due to its ability to sequester Zn and Mn. We present evidence that TdfH confers resistance to calprotectin and that TdfH facilitates gonococcal calprotectin binding and Zn accumulation in the presence or absence of calprotectin. Finally, we demonstrate that TdfH expression enhances N. gonorrhoeae NET survival. These studies identify for the first time a novel gonococcal defense strategy to host-mediated nutritional immunity, in which N. gonorrhoeae, via the TdT TdfH, utilizes calprotectin as a Zn source neutralizing its antimicrobial activity. These studies have yielded novel insights into the function and regulation of TdfG, TdfH and TdfJ in N. gonorrhoeae and have laid the framework for future investigation of TdT-mediated Zn acquisition and its role in bacterial pathogenesis.

Neisseria gonorrhoeae, the causative agent of the STI gonorrhea, is not preventable by vaccination and is rapidly developing resistance to antibiotics. One important strategy for gonococcal survival in the host is iron acquisition in the face of nutritional immunity. To overcome iron limitation, the gonococcus expresses TonB dependent transporters (TdTs), outer membrane proteins that facilitate nutrient acquisition. Of the TdTs, the transferrin (Tf), lactoferrin (Lf), and hemoglobin (Hb) receptors hijack iron directly from host proteins, and studies have already shown that the Tf receptor is essential for the initiation of human infection. Given that the TdTs are virulence factors, they are widely conserved across strains, and are not subject to antigenic variation, they are ideal targets for novel therapeutics and vaccine development. As such, studies exploring these proteins and their potential as vaccine candidates and antimicrobial targets are needed. In this study we report that loops of the Tf receptor protein TbpA are not strongly immunogenic, and the antibodies raised against them are incapable of inhibiting TbpA-Tf interactions on the gonococcal cell surface. We also report that the loop 3 helix motif of TbpA is a critical functional domain for Tf-binding and iron uptake; however, no single residue was identified that was essential for these functions. In addition, we report the development of a platform for the structure-function analysis of HpuA, a member of the poorly studied Hb receptor. We also present evidence that novel small molecules may be able to inhibit TbpA-Tf interaction, presenting the Tf receptor as a novel, species-specific antimicrobial target. Finally, we demonstrated that a novel drug, OSU-03012, has antimicrobial activity against the gonococcus through down-regulation of DnaK, a protein chaperone. These findings suggest that DnaK, a widely conserved protein, may be a universal target for antimicrobial development. These studies provide insight into the structure function relationship of TbpA, the drug potential of DnaK, and lay the framework for future investigations of the TdTs for use in a multi-antigen vaccine.

Helicobacter pylori colonizes the human stomach, where it causes gastritis that may develop into peptic ulcer disease or cancer when left untreated. Neisseria gonorrhoeae colonizes the urogenital tract and causes the sexually transmitted disease gonorrhea. In contrast, Lactobacillus species are part of the human microbiota, which is the resident microbial community, and are considered to be beneficial for health. The first host cell types that bacteria encounter when they enter the body are epithelial cells, which form the border between the inside and the outside, and macrophages, which are immune cells that engulf unwanted material.       The focus of this thesis has been the interaction between the host and bacteria, aiming to increase our knowledge of the molecular mechanisms that underlie the host responses and their effects on bacterial pathogenicity. Understanding the interactions between bacteria and the host will hopefully enable the development of new strategies for the treatment of infectious disease. In paper I, we investigated the effect of N. gonorrhoeae on the growth factor amphiregulin in cervical epithelial cells and found that the processing and release of amphiregulin changes upon infection. In paper II, we examined the expression of the transcription factor early growth response-1 (EGR1) in epithelial cells during bacterial colonization. We demonstrated that EGR1 is rapidly upregulated by many different bacteria. This upregulation is independent of the pathogenicity, Gram-staining type and level of adherence of the bacteria, but generally requires viable bacteria and contact with the host cell. The induction of EGR1 is mediated primarily by signaling through EGFR, ERK1/2 and β1-integrins. In paper III, we described the interactions of the uncharacterized protein JHP0290, which is secreted by H. pylori, with host cells. JHP0290 is able to bind to several cell types and induces apoptosis and TNF release in macrophages. For both of these responses, signaling through Src family kinases and ERK is essential. Apoptosis is partially mediated by TNF release. Finally, in paper IV, we showed that certain Lactobacillus strains can reduce the colonization of H. pylori on gastric epithelial cells. Lactobacilli decrease the gene expression of SabA and thereby inhibit the binding mediated by this adhesin. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 4: Manuscript.</p>

Carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) are exploited by human-specific pathogens to anchor themselves to or invade host cells. Interestingly, human granulocytes express a specific isoform, CEACAM3, that can direct efficient, opsonin-independent phagocytosis of CEACAM-binding Neisseria, Moraxella and Haemophilus species. As opsonin-independent phagocytosis of CEACAM-binding Neisseria depends on Src-family protein tyrosine kinase (PTK) phosphorylation of the CEACAM3 cytoplasmic domain, we hypothesized that an SH2-containing protein might be involved in CEACAM3-initiated, phagocytosis-promoting signals. Accordingly, we screened glutathione-S-transferase (GST) fusion proteins containing SH2 domains derived from a panel of signaling and adapter molecules for their ability to associate with CEACAM3. In vitro pull-down assays demonstrated that the SH2 domain of the adapter molecule Nck (GST-Nck SH2), but not other SH2 domains such as the Grb2 SH2 domain, interact with CEACAM3 in a phosphotyrosine-dependent manner. Either deletion of the cytoplasmic tail of CEACAM3, or point-mutation of a critical arginine residue in the SH2 domain of Nck (GST-NckSH2R308K) that disrupts phosphotyrosine binding, both abolished CEACAM3-Nck-SH2 interaction. Upon infection of human cells with CEACAM-binding Neisseria, full-length Nck comprising an SH2 and three SH3 domains co-localized with tyrosine phosphorylated CEACAM3 and associated bacteria as analyzed by immunofluorescence staining and confocal microscopy. In addition, Nck could be detected in CEACAM3 immunoprecipitates confirming the interaction in vivo. Importantly, overexpression of a GFP-fusion protein of the isolated Nck SH2 domain (GFP-Nck-SH2), but not GFP or GFP-Nck SH2 R308K reduced CEACAM3-mediated phagocytosis of CEACAM-binding Neisseria suggesting that the adaptor molecule Nck plays an important role in CEACAM3-initiated signaling leading to internalization and elimination of human-specific pathogens.