Submitted by Marlene Santos (marlene.bc.ufg@gmail.com) on 2014-10-02T19:41:10Z No. of bitstreams: 2 Dissertação - Liliam Borges Fernades - 2013.pdf: 1021436 bytes, checksum: ed17067b456cf27093e83d9cf999cd72 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Jaqueline Silva (jtas29@gmail.com) on 2014-10-02T20:11:13Z (GMT) No. of bitstreams: 2 Dissertação - Liliam Borges Fernades - 2013.pdf: 1021436 bytes, checksum: ed17067b456cf27093e83d9cf999cd72 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2014-10-02T20:11:13Z (GMT). No. of bitstreams: 2 Dissertação - Liliam Borges Fernades - 2013.pdf: 1021436 bytes, checksum: ed17067b456cf27093e83d9cf999cd72 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2013-06-27 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / It is estimated that between 60 and 80 million people worldwide enfretem difficulties to reproduce at some point in their lives and it is estimated that this ratio reaches approximately 20% of couples of reproductive age. Gonorrhea is a common bacterial infection caused by Neisseria gonorrhoeae (NG), transmitted almost exclusively by sexual or perianal contact. In women genital infection can be asymptomatic and when it discovers there may be serious complications such as pelvic inflammatory disease, and ectopic pregnancy, a leading cause of female infertility. The molecular diagnostic techniques are powerful allies in the tracking of NG and other sexually transmitted diseases (STD). Therefore, an investigation was conducted in the scientific literature in the form of a Systematic Literature Review (SLR) on the prevalence of NG, molecular diagnostic techniques and the samples used in the detection of this bacterium. Simultaneously, we developed a chart review in the last 4 years to detect the prevalence of gonococcal infection among other diagnoses as the cause of infertility in women seen at the Laboratory of Human Reproduction - Clinical Hospital, Goiânia, Goiás. SLR was found in 720 articles of which 694 were excluded due to previously established criteria and 26 were included in this review. The population was predominantly young 7 (27%) and female sex workers 6 (23%). The age ranged from 12 to 59 years. The more frequent biological material was endocervical swab 10 (38.46%), vaginal swabs 8 (30.76%), urine 4 (15.38%) and the association of these 3 (11.53%). The in-house PCR was the choice of 12 trials for the diagnosis of NG. In the other 14 studies different commercial diagnostic kits were used. Gradually more it is observed the use of biological samples collected from a less invasive diagnosis of STD. Molecular techniques found in this study are the most used in asymptomatic patients, since they have greater efficiency in the detection of NG, whose gold standard applies only in symptomatic patients. In reviewing the records among the 340 women studied, the prevalence of NG 0.6% and co-infection with Chlamydia trachomatis (CT) was 0.6%. DIP was present in 2.1% of cases and 2 cases of coinfection of NG and CT and the mean age was 33 years. The CT and NG infections had low prevalence in the studied sample and studies are needed in other breeding centers in the country to confirm the prevalence of infections in this particular group of women with infertility problems. / Estima-se que entre 60 e 80 milhões de pessoas em todo o mundo em algum momento de suas vidas enfrentem dificuldades para se reproduzirem e calcula-se que este índice atinja aproximadamente 20% dos casais em idade reprodutiva. A gonorréia é uma infecção bacteriana frequente, causada por Neisseria gonorrhoeae (NG), transmitida quase que exclusivamente por contato sexual ou perianal. Nas mulheres a infecção genital pode ser assintomática e quando se descobre pode haver complicações sérias como a doença inflamatória pélvica (DIP), uma das principais causas da infertilidade feminina e da gravidez ectópica. As técnicas diagnósticas moleculares são grandes aliadas no rastreamento de NG e outras doenças sexualmente transmissíveis (DST). Objetivos: realizar uma investigação na literatura científica no formato de uma Revisão Sistemática de Literatura (RSL) sobre a prevalência de NG em mulheres assintomáticas, observando a faixa etária, as técnicas moleculares diagnósticas e as amostras utilizadas na detecção dessa bactéria; e, uma revisão de prontuários dos últimos 4 anos para detectar a prevalência da infecção gonocócica entre outros diagnósticos como fator causal da infertilidade em mulheres atendidas no Laboratório de Reprodução Humana – Hospital das Clínicas/UFG, Goiânia, Goiás. Na RSL foram encontrados 720 artigos dentre os quais 694 foram excluídos por critérios previamente estabelecidos e 26 incluídos nesta revisão. A população predominante foi de jovens 7 (27%) e mulheres profissionais do sexo 6 (23%). A idade variou de 12 a 59 anos. O material biológico mais frequente foi esfregaço endocervical 10 (38,46%), esfregaço vaginal 8 (30,76%), urina 4(15,38%) e a associação dos mesmos 3(11,53%). A técnica PCR convencional foi a técnica de escolha em 12 estudos para diagnóstico de NG. Nos outros 14 estudos foram utilizados distintos kits diagnósticos comerciais. Cada vez mais se observa o uso de amostras biológicas coletadas de forma menos invasiva para o diagnóstico de DST. As técnicas moleculares encontradas no presente estudo são as mais empregadas em pacientes assintomáticos, pois apresentam maior eficiência na detecção de NG, cujo padrão ouro só se aplica em pacientes sintomáticos. Na revisão de prontuários entre as 340 mulheres estudadas, a prevalência de NG de 0,6% e coinfecção com Chlamydia trachomatis (CT) foi 0,6%. DIP foi presente em 2,1% dos casos estudados sendo 2 casos por coinfecção de NG e CT e a média de idade foi 33 anos. As infecções por CT e NG tiveram baixa prevalência na amostra estudada e são necessários estudos em outros centros de reprodução do país para confirmar a prevalência de infecções nesse grupo particular de mulheres com problemas de infertilidade.

A gonorréia permanece até os dias atuais como um problema de saúde pública. A resistência plasmidial à penicilina, em cepas de Neisseria gonorrhoeae, constitui um problema crescente, preocupando autoridades sanitárias em todo o mundo. O conhecimento das características epidemiológicas e da dinâmica destas cepas na população é imperativo, para o sucesso de intervenções de combate e controle da gonorréia no Brasil e no mundo. Os fenômenos de instalação, manutenção e propagação da Neisseria gonorrhoeae produtora de penicilinase são ainda pouco conhecidos. Este trabalho estudou as características fenotípicas (comportamento de sensibilidade às drogas antimicrobianas, características do plasmídio de resistência, auxotipo e sorotipo), da Neisseria gonorrhoeae produtora de penicilinase (NGPP), objetivando fornecer conhecimentos quanto aos fenômenos de instalação, manutenção e propagação destas cepas, de forma a contribuir com o programa de combate e controle da gonorréia na cidade de São Paulo e no Brasil. Foram estudadas 15 cepas de Neisseria gonorrhoeae produtora de penicilinase (NGPP) e 50 de Neisseria gonorrhoeae não produtora penicilinase (não NGPP), isolados no serviço de Doenças Sexualmente Transmissíveis (DST) da Faculdade de Saúde Pública da Universidade de São Paulo, no período de abril de 1985 a abril de 1990. Com uma incidência de 3,11 por cento de cepas de NGPP, os resultados permitiram colaborar para formação de perfil epidemiológico das cepas produtoras de penicilinase na cidade de São Paulo. Quanto as características de sensibilidade aos antimicrobianos, foram observados altos níveis de resistência a tetraciclina. As demais características fenotípicas, plasmídios, auxotipo e sorotipo, demonstram que a cidade de São Paulo é uma região geográfica \"aberta\" ao fenômeno de \"importação\", tendo sido observadas características epidemiológicas descritas em várias outras localidades no mundo. Os resultados obtidos neste trabalho reforçam a necessidade da instalação de um programa de vigilância epidemiológica, visando manter sob controle a dinâmica da Neisseria gonorrhoeae, colaborando com o programa de combate e controle da gonorréia na cidade de São Paulo e no Brasil. / Gonorrhoea has been considered as a public health problem up to these days. The plasmid penicilin resistance, in Neisseria gonorrhoeae strains, is an increasing problem which concerns authorities from the whole world. The Knowledge on the epidemiological characteristics and on the dynamics of these strains in the populations is extremely important for the success of interventions and gonorrhoea contrai programmes in Brazil and in the world. The onset phenomena, maintenance and proliferation of the penicilin producing Neisseria gonorrhoeae (PPNG) are not well known yet. This study presents the phenotype characteristics (sensibility to antimicrobial drugs, plasmid resistance characteristics, auxotype and serotype) of the PPNG. The objectives of the study are to contribute to the knowledge related to the onset phenomena, maintenance and proliferation of these strains, so that we can contribute to the gonorrhoea contrai programme in the city of São Paulo and in Brazil. We studied 15 strains of penicilin producing Neisseria gonorrhoeae (PPNG) and 50 of non penicilin producing Neisseria gonorrhoeae (non PPNG), which were isolated in the Sexually Transmitted Disease Service of the Faculty of Public Health of the University of São Paulo, from April 1985 to April 1990. With an incidence rate of 3.11 per cent of Neisseria gonorrhoeae, the results allowed us to design an epidemiological profile of the penicilin producing strains in São Paulo. Regarding the antimicrobial sensibility characteristics, we observed high leveis of resistance to tetracycline. The other phenotype characteristics (plasmid, auxotype, and serotype) show that the city of São Paulo is a geographic region open to \"importation\" phenomenon, since we have observed epidemiologic charactéristics described in several places in the world. The results of this reinforce the necessity of setting up an epidemiological surveillance programme with the objective of controlling the Neisseria gonorrhoeae dynamics and, in this way, to colaborate with the gonorrhoea control programme in the city of São Paulo and in Brazil.

Chan, Lap Kwan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 131-147). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.i / ABSTRACT --- p.iii / ABSTRACT (CHINESE VERSION) --- p.v / TABLE OF CONTENTS --- p.vii / LIST OF TABLES --- p.xiv / LIST OF FIGURES --- p.xv / LIST OF ABBREVIATIONS --- p.xvii / Chapter CHAPTER 1 --- INTRODUCTION / Chapter 1.1 --- Background --- p.2 / Chapter 1.2 --- Prevalence of antimicrobial reisitance in gonococcal strains --- p.4 / Chapter 1.2.1 --- Prevalence of penicillin resistant gonococcal strains --- p.5 / Chapter 1.2.2 --- Prevalence of tetracycline resistant gonococcal strains --- p.6 / Chapter 1.2.3 --- Prevalence of quinolone resistant gonococcal strains. --- p.7 / Chapter 1.2.4 --- Emergence of generation cephalosporin reduced susceptible gonococcal strains --- p.8 / Chapter 1.2.5 --- Monitoring the prevalence of gonorrhea --- p.9 / Chapter 1.3 --- Gonococcal Antimicrobial Surveillance Program --- p.10 / Chapter 1.4 --- Antimicrobial Resistance Mechanisms in N. gonorrhoeae --- p.11 / Chapter 1.4.1 --- "Innate Resistance Mechanisms in N, gonorrhoeae" --- p.11 / Chapter 1.4.1.1 --- Natural mechanisms in N. gonorrhoeae against toxic substance --- p.11 / Chapter 1.4.1.2 --- Efflux pump inhibitors --- p.13 / Chapter 1.4.2 --- Development of acquired antimicrobial resistance in N. gonorrhoeae --- p.14 / Chapter 1.4.2.1 --- Penicillin --- p.14 / Chapter 1.4.2.1.1 --- Chromosomal-mediated resistance --- p.14 / Chapter 1.4.2.1.2 --- Plasmid-mediated resistance --- p.16 / Chapter 1.4.2.2 --- Tetracycline --- p.16 / Chapter 1.4.2.2.1 --- Plasmid-mediated resistance --- p.17 / Chapter 1.4.2.2.2 --- Chromosomal mediated resistance --- p.18 / Chapter 1.4.2.3 --- Fluroquinolone --- p.18 / Chapter 1.4.2.3.1 --- Resistant mechanism in quinolone resistant gonococcal strains --- p.19 / Chapter 1.4.2.3.1.1 --- gyrA andparC --- p.19 / Chapter 1.4.2.3.1.2 --- NorM efflux system --- p.21 / Chapter 1.4.2.4 --- 3rd generation cephalosporins --- p.22 / Chapter 1.4.2.4.1 --- Mosaic penA structure in reduced susceptible gonococcal strains --- p.22 / Chapter 1.4.2.4.2 --- Other mechanisms related to reduced susceptibility in gonococcal strains --- p.24 / Chapter 1.5 --- Application of molecular typing methods to study the epidemiology of N. gonorrhoeae --- p.24 / Chapter 1.5.1 --- Opa typing --- p.25 / Chapter 1.5.2 --- K gonorrhoeae Multi-Antigen Sequence Typing --- p.25 / Chapter 1.6 --- Project Objectives --- p.27 / Chapter CHAPTER 2. --- MATERIALS AND METHODS / Chapter 2.1 --- Collecting gonococcal strains --- p.29 / Chapter 2.2 --- Culturing of N. gonorrhoeae --- p.29 / Chapter 2.3 --- Identification --- p.29 / Chapter 2.3.1 --- Gram staining test --- p.30 / Chapter 2.3.2 --- Oxidase test --- p.31 / Chapter 2.3.3 --- Cabohydrate utilization test --- p.31 / Chapter 2.4 --- In-vitro antimicrobial susceptibility testing --- p.32 / Chapter 2.4.1 --- Preparation of cell cultures for MIC tests --- p.32 / Chapter 2.4.2 --- Preparation of antimicrobial agents for MIC tests --- p.32 / Chapter 2.4.3 --- Inoculum preparation and delivering --- p.33 / Chapter 2.5 --- Preparation of genomic DNA for detection of mutations --- p.34 / Chapter 2.6 --- Study of Resistant Mechanism against fluoroquinolone --- p.34 / Chapter 2.6.1 --- PGR detection of mutations in gyrA and parC genes --- p.35 / Chapter 2.6.2 --- Optimization of gyrA and parC genes PGR --- p.35 / Chapter 2.6.3 --- Detection of PGR products for gyrA and parC genes --- p.37 / Chapter 2.6.4 --- Purification of Amplified DNA products --- p.37 / Chapter 2.7 --- Tests of efflux inhibitor on N. gonorrhoeae --- p.38 / Chapter 2.7.1 --- Effect ofCCCP --- p.39 / Chapter 2.7.2 --- Effect of Reserpine --- p.39 / Chapter 2.8 --- Study of Resistant mechanism against β-lactams --- p.40 / Chapter 2.8.1. --- Detection for the presence of β-lactamase --- p.40 / Chapter 2.8.2 --- Mosaic penA patterns --- p.40 / Chapter 2.8.2.1 --- Detection of mutations in penA gene --- p.40 / Chapter 2.8.2.2 --- Optimization of penA gene PGR --- p.41 / Chapter 2.8.2.3 --- Detection of PGR products --- p.43 / Chapter 2.8.2.4 --- Purification of Amplified DNA products --- p.44 / Chapter 2.9 --- Detection of the presence of tetM determinant --- p.45 / Chapter 2.9.1 --- Optimization of tetM determinant PCRs --- p.46 / Chapter 2.9.2 --- Detection of PGR products --- p.47 / Chapter 2.10 --- Detection of different allele types in tbpB andpor genes --- p.48 / Chapter 2.10.1 --- Optimization of PGR for NG-MAST --- p.48 / Chapter 2.10.2 --- Detection of PCR products --- p.49 / Chapter 2.10.3 --- PCR product purification --- p.50 / Chapter 2.11 --- Sequencing of the PCR products --- p.51 / Chapter 2.12 --- Data Analysis --- p.52 / Chapter CHAPTER 3. --- RESULTS / Chapter 3.1 --- Gonococcal strains collected --- p.55 / Chapter 3.2 --- Identification of gonococcal strains --- p.55 / Chapter 3.3 --- MIC of Antimicrobial agents --- p.56 / Chapter 3.3.1 --- Interpretive Criteria --- p.56 / Chapter 3.3.2 --- Ciprofloxacin --- p.56 / Chapter 3.3.3 --- Penicillin --- p.57 / Chapter 3.3.4 --- Tetracycline --- p.57 / Chapter 3.3.5 --- Ceftriaxone --- p.58 / Chapter 3.3.6 --- Cefixime --- p.58 / Chapter 3.3.7 --- Cefotaxime --- p.58 / Chapter 3.3.8 --- Spectinomycin --- p.58 / Chapter 3.3.9 --- Levofloxacin --- p.59 / Chapter 3.3.10 --- Ceftibuten --- p.59 / Chapter 3.4 --- Result of PGR --- p.60 / Chapter 3.4.1 --- gyrA andparC genes --- p.60 / Chapter 3.4.2 --- penA gene --- p.61 / Chapter 3.4.3 --- tbpB and por genes --- p.62 / Chapter 3.4.4 --- tetM determinant --- p.62 / Chapter 3.5 --- β-lactamase --- p.63 / Chapter 3.6 --- Efflux pump inhibitor --- p.63 / Chapter 3.6.1 --- CCCP --- p.64 / Chapter 3.6.2 --- Reserpine --- p.64 / Chapter 3.7 --- Detection of Mutations --- p.66 / Chapter 3.7.1 --- gyrA and parC genes --- p.66 / Chapter 3.7.2 --- penA gene --- p.68 / Chapter 3.8 --- NG-MAST --- p.70 / Chapter 3.8.1 --- tbpB and por gene --- p.71 / Chapter 3.9 --- Porin mutation --- p.72 / Chapter CHAPTER 4. --- DISCUSSION / Chapter 4.1 --- Sampling --- p.74 / Chapter 4.2 --- Methodology --- p.75 / Chapter 4.3 --- MIC distribution of different antimicrobial agents --- p.76 / Chapter 4.4 --- Mechanisms of quinolone resistance --- p.78 / Chapter 4.4.1 --- Mutations at QRDRs --- p.78 / Chapter 4.4.2 --- Association of the number of mutations at parC gene with MIC levels against fluroquinolones --- p.80 / Chapter 4.5 --- Penicillin and Tetracycline Resistant Mechanisms --- p.81 / Chapter 4.6 --- Efflux system --- p.85 / Chapter 4.7 --- NG-MAST --- p.88 / Chapter 4.8 --- Mosaic penA pattern --- p.89 / Chapter 4.9 --- Management of gonorrhea --- p.90 / Chapter CHAPTER 5. --- CONCLUSIONS / REFERENCES

A gonorréia permanece até os dias atuais como um problema de saúde pública. A resistência plasmidial à penicilina, em cepas de Neisseria gonorrhoeae, constitui um problema crescente, preocupando autoridades sanitárias em todo o mundo. O conhecimento das características epidemiológicas e da dinâmica destas cepas na população é imperativo, para o sucesso de intervenções de combate e controle da gonorréia no Brasil e no mundo. Os fenômenos de instalação, manutenção e propagação da Neisseria gonorrhoeae produtora de penicilinase são ainda pouco conhecidos. Este trabalho estudou as características fenotípicas (comportamento de sensibilidade às drogas antimicrobianas, características do plasmídio de resistência, auxotipo e sorotipo), da Neisseria gonorrhoeae produtora de penicilinase (NGPP), objetivando fornecer conhecimentos quanto aos fenômenos de instalação, manutenção e propagação destas cepas, de forma a contribuir com o programa de combate e controle da gonorréia na cidade de São Paulo e no Brasil. Foram estudadas 15 cepas de Neisseria gonorrhoeae produtora de penicilinase (NGPP) e 50 de Neisseria gonorrhoeae não produtora penicilinase (não NGPP), isolados no serviço de Doenças Sexualmente Transmissíveis (DST) da Faculdade de Saúde Pública da Universidade de São Paulo, no período de abril de 1985 a abril de 1990. Com uma incidência de 3,11 por cento de cepas de NGPP, os resultados permitiram colaborar para formação de perfil epidemiológico das cepas produtoras de penicilinase na cidade de São Paulo. Quanto as características de sensibilidade aos antimicrobianos, foram observados altos níveis de resistência a tetraciclina. As demais características fenotípicas, plasmídios, auxotipo e sorotipo, demonstram que a cidade de São Paulo é uma região geográfica \"aberta\" ao fenômeno de \"importação\", tendo sido observadas características epidemiológicas descritas em várias outras localidades no mundo. Os resultados obtidos neste trabalho reforçam a necessidade da instalação de um programa de vigilância epidemiológica, visando manter sob controle a dinâmica da Neisseria gonorrhoeae, colaborando com o programa de combate e controle da gonorréia na cidade de São Paulo e no Brasil. / Gonorrhoea has been considered as a public health problem up to these days. The plasmid penicilin resistance, in Neisseria gonorrhoeae strains, is an increasing problem which concerns authorities from the whole world. The Knowledge on the epidemiological characteristics and on the dynamics of these strains in the populations is extremely important for the success of interventions and gonorrhoea contrai programmes in Brazil and in the world. The onset phenomena, maintenance and proliferation of the penicilin producing Neisseria gonorrhoeae (PPNG) are not well known yet. This study presents the phenotype characteristics (sensibility to antimicrobial drugs, plasmid resistance characteristics, auxotype and serotype) of the PPNG. The objectives of the study are to contribute to the knowledge related to the onset phenomena, maintenance and proliferation of these strains, so that we can contribute to the gonorrhoea contrai programme in the city of São Paulo and in Brazil. We studied 15 strains of penicilin producing Neisseria gonorrhoeae (PPNG) and 50 of non penicilin producing Neisseria gonorrhoeae (non PPNG), which were isolated in the Sexually Transmitted Disease Service of the Faculty of Public Health of the University of São Paulo, from April 1985 to April 1990. With an incidence rate of 3.11 per cent of Neisseria gonorrhoeae, the results allowed us to design an epidemiological profile of the penicilin producing strains in São Paulo. Regarding the antimicrobial sensibility characteristics, we observed high leveis of resistance to tetracycline. The other phenotype characteristics (plasmid, auxotype, and serotype) show that the city of São Paulo is a geographic region open to \"importation\" phenomenon, since we have observed epidemiologic charactéristics described in several places in the world. The results of this reinforce the necessity of setting up an epidemiological surveillance programme with the objective of controlling the Neisseria gonorrhoeae dynamics and, in this way, to colaborate with the gonorrhoea control programme in the city of São Paulo and in Brazil.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

Neisseria gonorrhoeae is an obligate human pathogen that causes the common sexually- transmitted infection, gonorrhea. Gonococcal infections cause significant morbidity, particularly among women, as the organism ascends to the upper reproductive tract, resulting in pelvic inflammatory disease, ectopic pregnancy and infertility. Antibiotic resistance rates have risen dramatically, leading to severe restriction of treatment options for gonococcal disease. Gonococcal infections do not elicit protective immunity nor is there an effective vaccine to prevent the disease. Thus, further characterization of expression, function and regulation of surface antigens could lead to better treatment and prevention modalities in the future. N. gonorrhoeae express a repertoire of TonB-dependent transporters for the acquisition of iron. All of these transporters are under the transcriptional regulation of Fur. We investigated putative intracellular iron sources utilized by gonococci and the role that the TonB-dependent transporter, TdfF, played in this acquisition. We determined that ascorbate which could prevent ferritin degradation or withhold iron from gonococci, inhibited intracellular survival. The utilization of iron from the iron binding moiety 2, 5-DHBA of the putative mammalian siderophore was also examined. In this study we continued to investigate the regulation of TdfF and further investigate potential host-specific inducing molecules for TdfF expression. We investigated the regulation of tdfF expression and the role of MpeR, an AraC-like regulator, in tdfF expression. We determined that MpeR, interacted specifically with the DNA sequence upstream of fetA and activated FetA expression. We confirmed that the outer membrane transporter, FetA, allows gonococcal strain FA1090 to utilize the xenosiderophore, ferric-enterobactin, as an iron source. However, we further demonstrated that FetA has an extended range of substrates that encompasses other catecholate xenosiderophores, including ferric-salmochelin and the dimers and trimers of dihydroxybenzoylserine. We demonstrated that fetA is encoded as part of an iron-repressed, MpeR-activated operon, which putatively encodes other iron transport proteins. These iron transport proteins also play a role in xenosiderophore acquisition. We also identified genetic differences that may explain why some gonococcal strains are capable of xenosiderophore internalization in a TonB-dependent pathway and other strains are restricted to TonB-independent pathways. Interestingly, the chromosomal locus that codes for mpeR and tdfF is pathogen specific. Thus understanding more about the TonB-dependent transporter and AraC-like regulator may further elucidate the pathogenicity of N gonorrhoeae.

The neisserial transferrin binding proteins (Tbps) comprise a bipartite system for iron acquisition from human transferrin. TbpA is a TonB-dependent outer membrane protein that forms the pore for iron internalization. TbpB is a surface-exposed lipoprotein that makes the iron-uptake process more efficient. Previous studies have shown that the genes encoding these proteins are arranged in a bicistronic operon, with the tbpB gene located upstream of tbpA. The operon is under the control of the ferric uptake regulator (Fur) protein. However, promoter elements necessary for the regulation of the operon have not been experimentally defined. In this study, putative regulatory motifs were confirmed by mutagenesis. Further examination of the sequence upstream of these promoter/operator motifs led to the identification of two direct repeats. We hypothesized that these repeats may be involved in further regulation of the operon. Insertional mutagenesis of the repeats resulted in altered transcript and protein levels. These results confirmed that the region upstream of the operon serves as an extended regulatory region. A comprehensive investigation of the expression of the operon in response to different environmental stimuli that gonococci might encounter upon infection was also conducted. Changes in osmolarity, carbon source, cAMP availability, and H2O2 stress did not alter expression of the operon at the transcript or protein levels. However, low oxygen levels resulted in decreased tbpBA transcript and protein. These results are biologically relevant, and provide new insights into the use of the transferrin binding proteins as vaccine candidates. Lastly, the role of G4 DNA sequences identified in the vicinity of the tbpBA operon was investigated. We hypothesized that G4 DNA structures could be involved in the regulation of the operon. Results presented here indicate that interference with these sequences appears to have no effect on expression of the operon. However, identification of potential G4-forming sequences in the non-coding regions upstream and downstream of the operon suggests their importance, perhaps in mediating recombination which could lead to increased antigenic diversity.

Several bacterial pathogens persist and survive in the host by modulating host cell death pathways. Previous studies have demonstrated that the sexually transmitted pathogen, Neisseria gonorrhoeae, can induce or inhibit host cell death. N. gonorrhoeae is a mucosal pathogen and, in females, initiates infection in epithelial cells of the ectocervix and endocervix. Mucosal epithelial cells of the female genital tract are the first line of defense, and thus their cellular fate can alter the early immune response to invading pathogens such as N. gonorrhoeae. The mechanisms by which N. gonorrhoeae modulates cell death are not clear, although a role for the inhibitor of apoptosis-2 (cIAP2) has been proposed. In the present study, we demonstrate that N. gonorrhoeae stimulation induces a transient increase in cIAP2 protein levels in human cervical epithelial cells. High intracellular protein levels were observed during early N. gonorrhoeae stimulation and were followed by a marked intracellular decrease at 24 h. At this time point, we observed increased levels of extracellular cIAP2 associated with exosomes, which are nano-sized vesicles that carry protein and coding RNA as cargo from one cell to another. We also observed that depletion of cIAP2 in N. gonorrhoeae stimulated cells resulted in cell death resembling necroptosis, an inflammatory form of cell death. Furthermore, inhibition of cIAP2 led to an increase in interleukin-1β production. Exosomes have been found to have important roles in cell communication during microbial infection. Here, we demonstrate that N. gonorrhoeae induces exosome production and alters exosome content. We also demonstrate that exosomes elicit cytokine responses in uninfected naïve cells. Collectively, these studies highlight an additional mechanism for epithelial cells to orchestrate the immune response in the female genital tract during N. gonorrhoeae infection.

Neisseria gonorrhoeae is a strict human pathogen and the causative agent of the sexually transmitted disease, gonorrhea. Gonococcal (GC) diseases remain one of the most reported sexually transmitted infections (STI) worldwide, representing a significant threat to reproductive health and burden on global health systems, accounting for 541,987 disability adjusted-life years in the year 2011. Infection by N. gonorrhoeae also increases the likelihood of patient acquisition and transmission of human immunodeficiency virus (HIV). Unfortunately, antibiotic treatment to gonococcal diseases is being threatened by the rapid spread of resistance to third-generation cephalosporins, the remaining treatment option used in clinics. The urgency of the situation is compounded by the relative lack of immunological protection conferred by previous infection by the bacterium. In response to the emergence of multidrug resistance, renewed energies are being directed towards the development of an effective, broadly protective vaccine. Difficulties in vaccine development arise from a lack of known correlates of protective immunity; there is no known broadly cross-protective immunity to GC and a truly reflective animal model has not been available. Nonetheless, previous studies have indicated that porins, neisserial major outer membrane proteins, are promising vaccine candidates. PorB makes up over 60% of the bacterium's outer membrane content and is involved in solute and ion exchange, invasion of target host cells, and evasion of host immunity. Porins from both the gonococcus and the meningococcus have been shown to have immunostimulatory activity, boosting B and dendritic (DC) cell proliferation and maturation in the absence of an exogenous adjuvant as mediated by TLR2 and MyD88. Importantly, as a potential vaccine candidate, PorB has relatively low antigenic variability, and can induce bactericidal antibodies. Gonococcal PorB was purified from a genetically modified strain, MS11delP3, which lacks another outer membrane protein, RMP, which is known to induce bactericidal blocking antibodies. PorB was formed into pure protein micelles, termed proteosomes, to protect the integrity of the native trimeric structure. Our study demonstrated that gonococcal PorB is able to stimulate both human embryonic kidney (HEK) cell line that overexpresses TLR2 and mouse primary macrophages (similar to the meningococcal PorB). To test PorB's immunogenicity, mice were immunized three times at two week intervals with PorB and porin specific IgG levels were measured. Unfortunately, PorB elicited lower levels of porin specific IgG than what was expected, which may be due to technical issues. We are currently investigating various possibilities. In addition, further immunization studies shall be carried out to better contextualize these results.

The obligate human pathogen Neisseria gonorrhoeae is responsible for the widespread sexually transmitted disease gonorrhoea, which in rare cases also leads to the development of disseminated gonococcal infection (DGI). DGI is mediated by PorBIA-expressing bacteria that invade host cells under low phosphate condition by interaction with the scavenger receptor-1 (SREC-I) expressed on the surface of endothelial cells. The interaction of PorBIA and SREC-I was analysed using different in vitro approaches, including surface plasmon resonance experiments that revealed a direct phosphate-independent high affinity interaction of SREC-I to PorBIA. However, the same binding affinity was also found for the other allele PorBIB, which indicates unspecific binding and suggests that the applied methods were unsuitable for this interaction analysis. Since N. gonorrhoeae was recently classified as a “super-bug” due to a rising number of antibiotic-resistant strains, this study aimed to discover inhibitors against the PorBIA-mediated invasion of N. gonorrhoeae. Additionally, inhibitors were searched against the human pathogen Chlamydia trachomatis, which causes sexually transmitted infections as well as infections of the upper inner eyelid. 68 compounds, including plant-derived small molecules, extracts or pure compounds of marine sponges or sponge-associated bacteria and pipecolic acid derivatives, were screened using an automated microscopy based approach. No active substances against N. gonorrhoeae could be identified, while seven highly antichlamydial compounds were detected. The pipecolic acid derivatives were synthesized as potential inhibitors of the virulence-associated “macrophage infectivity potentiator” (MIP), which exhibits a peptidyl prolyl cis-trans isomerase (PPIase) enzyme activity. This study investigated the role of C. trachomatis and N. gonorrhoeae MIP during infection. The two inhibitors PipN3 and PipN4 decreased the PPIase activity of recombinant chlamydial and neisserial MIP in a dose-dependent manner. Both compounds affected the chlamydial growth and development in epithelial cells. Furthermore, this work demonstrated the contribution of MIP to a prolonged survival of N. gonorrhoeae in the presence of neutrophils, which was significantly reduced in the presence of PipN3 and PipN4. SF2446A2 was one of the compounds that had a severe effect on the growth and development of C. trachomatis. The analysis of the mode of action of SF2446A2 revealed an inhibitory effect of the compound on the mitochondrial respiration and mitochondrial ATP production of the host cell. However, the chlamydial development was independent of proper functional mitochondria, which excluded the connection of the antichlamydial properties of SF2446A2 with its inhibition of the respiratory chain. Only the depletion of cellular ATP by blocking glycolysis and mitochondrial respiratory chain inhibited the chlamydial growth. A direct effect of SF2446A2 on C. trachomatis was assumed, since the growth of the bacteria N. gonorrhoeae and Staphylococcus aureus was also affected by the compound. In summary, this study identified the severe antichlamydial activity of plant-derived naphthoquinones and the compounds derived from marine sponges or sponge-associated bacteria SF2446A2, ageloline A and gelliusterol E. Furthermore, the work points out the importance of the MIP proteins during infection and presents pipecolic acid derivatives as novel antimicrobials against N. gonorrhoeae and C. trachomatis. / Neisseria gonorrhoeae ist ein obligat humanpathogenes Bakterium, das für die weltweit verbreitete sexuell übertragbare Krankheit Gonorrhoe verantwortlich ist. In seltenen Fällen kann es auch zur Ausbildung der Disseminierten Gonokokken-Infektion (DGI) kommen, die mit der Expression des Gonokokken Oberflächenproteins PorBIA assoziiert ist. PorBIA-exprimierende Bakterien invadieren in die Wirtszelle unter phosphatfreien Bedingungen, was durch eine Interaktion mit dem zellulären Oberflächenrezeptor scavenger receptor-1 (SREC-I) vermittelt wird. Die direkte Interaktion zwischen PorBIA und SREC-I wurde mittels verschiedenster Methoden analysiert, einschließlich einer Oberflächenplasmonresonanz-analyse, die eine direkte Bindung von PorBIA zu SREC-I in einem phosphatunabhängigen Schritt aufzeigte. Allerdings wurde dieselbe Affinität auch zu PorBIB gefunden, was auf eine unspezifische Bindung hindeutet und dafür spricht, dass die verwendeten Methoden für diese Interaktionsanalyse ungeeignet sind. N. gonorrheae wurde vor kurzem wegen der stetig steigenden Anzahl antibiotikaresistenter Stämme als „Superkeim“ bezeichnet. Aufgrund dessen wurden Inhibitoren gegen die PorBIA-vermittelte Invasion von N. gonorrhoeae, aber auch gegen Chlamydia trachomatis, den humanpathogenen Erreger von sexuell übertragbaren Infektionen und chronisch-follikulärer Bindehautentzündung, gesucht. 68 niedermolekulare Substanzen wurden mittels eines automatisierten Fluoreszenzmikroskopieverfahrens auf ihre inhibitorische Wirkung hin analysiert. Zu den getesteten Substanzen zählten pflanzenabstammende Stoffe, Isolate aus marinen Schwämmen oder Schwamm-assoziierten Bakterien, sowie Pipecolinsäure-Derivate. Gegen N. gonorrheae konnten keine Substanzen identifiziert werden, während sieben antichlamydiale Inhibitoren detektiert wurden. Pipecolinsäurederivate wurden synthetisiert als potentielle Inhibitoren des virulenz-assoziierten Proteins “macrophage infectivity potentiator” (MIP), das eine Peptidyl-Prolyl-cis-trans-Isomerase Aktivität besitzt. Diese Arbeit untersuchte die Rolle des MIP Proteins von N. gonorrhoeae und C. trachomatis während einer Infektion. Die zwei Inhibitoren PipN3 und PipN4 senkten die PPIase Aktivität des rekombinanten Chlamydien und Neisserien MIPs. Beide Substanzen beeinträchtigten das chlamydiale Wachstum und die Entwicklung in Epithelzellen. Ebenso konnte eine tragende Rolle des N. gonorrhoeae MIPs für das Überleben der Bakterien in Gegenwart von Neutrophilen aufgezeigt werden, das durch PipN3 und PipN4 inhibiert wurde. SF2446A2 war einer der Inhibitoren, der einen erheblichen Effekt auf das Wachstum und die Entwicklung von C. trachomatis aufgewiesen hat. Während der Analyse des Wirkmechanismus von SF2446A2 konnte eine Hemmung der mitochondrialen Atmungskette und eine Abnahme der mitochondrialen ATP Produktion in der Wirtszelle festgestellt werden. Allerdings war die Entwicklung von C. trachomatis unabhängig von der Funktionsfähigkeit der Mitochondrien. Eine Verbindung zwischen der antichlamydialen Wirkung von SF2446A2 und der Inhibierung der Mitochondrienatmungskette konnte damit ausgeschlossen werden. Nur das Reduzieren von zellulärem ATP durch Blockieren der Glykolyse und mitochondrialen Atmungskette verursachte eine Beeinträchtigung des Chlamydienwachstums. Eine direkte Auswirkung von SF2446A2 auf C. trachomatis wurde angenommen, da die Substanz auch das Wachstum von anderen Bakterien wie N. gonorrhoeae und Staphylococcus aureus inhibierte. Zusammengefasst identifizierte diese Studie die antichlamydiale Aktivität pflanzenabstammender Naphthochinone und der Isolate aus marinen Schwämmen oder Schwamm-assoziierten Bakterien SF2446A2, ageloline A und gelliusterol E. Ebenso verdeutlicht die Arbeit die Bedeutung der MIP Proteine während der Infektion und legt Pipecolinsäurederivate als mögliche neue Antibiotika gegen N. gonorrhoeae und C. trachomatis nahe.