Submitted by Priscila Nascimento (pnascimento@icict.fiocruz.br) on 2012-12-10T12:32:54Z No. of bitstreams: 1 iaralice-souza.pdf: 1338553 bytes, checksum: 59fa26df56bce23aa1b5861c460f716f (MD5) / Made available in DSpace on 2012-12-10T12:32:54Z (GMT). No. of bitstreams: 1 iaralice-souza.pdf: 1338553 bytes, checksum: 59fa26df56bce23aa1b5861c460f716f (MD5) Previous issue date: 2001 / Fundação Oswaldo Cruz. Instituto de Tecnologia em Imunobiológicos. Rio de Janeiro, RJ, Brasil. / Neisseria meningitidisdo grupo C é uma bactéria encapsulada causadora dediversas doenças, está associada à altas taxas de mortalidade e portanto é de grande importância para a saúde pública. Bio-Manguinhos está desenvolvendo uma vacina conjugada formada pela ligação covalente do polissacarídeo capsular àanatoxina tetânica e esta vacina, atualmente, está sendo avaliada em estudos clínicosde Fase II em crianças de 1 a 9 anos. A quantificação de componentes livres como polissacarídeos e proteínas faz parte do controle de processo de vacinas conjugadas e tem o objetivo de evitar o aparecimento de reações adversas exacerbadas e/ou redução da imunogenicidade do componente vacinal. A Organização Mundial de Saúde preconiza níveis máximos de proteína livre no conjugado vacinal de 5%, mas não estabelece um limite máximo para o polissacarídeo livre para a vacina conjugada contra o grupo C. Desta forma, o objetivo deste estudo foi desenvolver e validar métodos de controle de qualidade adequados para separar e quantificar estes componentes livres presentes na vacina meningocócica C conjugada brasileira, utilizando a técnica de eletroforese capilar (EC). Para a separação da proteína livre foram comparados os modos de eletroforese capilar de zona livre (CZE) e cromatografia eletrocinética micelar (MEKC). Diferentes condições de migração da amostravariando-se parâmetros como pH, temperatura, tensão, concentração do tampão, ciclodextrinas e de surfactante dodecil sulfato de sódio (SDS) foram estudadas. Os resultados demonstraram que a melhor separação do conjugado foi obtida por MEKC utilizando tampão tetraborato de sódio (TBNa) 150 mM, 25 mM de SDS, 60°C, 30 kV e pH 9,3. Entretanto, nos modos de EC estudados não foi possível obter a separação completa dos componentes, sendo necessária a utilização de outro processo. Por outro lado, por CZE foi possível observar a separação da proteína ativada da nativa, demonstrando a necessidade de otimização da reação de ativação da proteína, a fim de aumentar orendimento da reação de conjugação. A separação completa do açúcar livre presente no conjugado foi obtida empregando CZE utilizando tampão TBNa 50 mM, 40°C, 30 kV e pH 10. Com as condições escolhidas foi possível determinar o conteúdo de polissacarídeo livre nos lotes de conjugado e validar o método proposto, que se mostrou linear na faixa de 0,047 a 0,164 mg/mL, apresentou efeito matriz, 0,0154 mg/mL de limite de detecção e 0,0454mg/mL de limite de quantificação. Após as etapas de validação, foram quantificados alguns lotes de conjugado e observou-se um alto teor de açúcar livre nos lotes com longo período de estocagem a 4°C. Desse modo, fez-se a avaliação de um lote recentemente produzido e obteve-se o valor de 19,08% de polissacarídeo livre. A fim de estimar o tempo de estocagem máximo do conjugado foram realizadas análises com 30, 60 e 90 dias de estocagem a 4°C. Os valores encontrados até 60 dias não foram significativamente diferentes dosdeterminados no tempo zero. No entanto, com 90 dias de estocagem ocorreu uma modificação do perfil do conjugado que impossibilitou a sua quantificação. A metodologia desenvolvida e validada será introduzida no controle de qualidade do lote de conjugado que será submetido aos estudos clínicos de Fase III e na rotina da vacina conjugada estudada. Além disto, o conhecimento adquirido poderá ser empregado no controle de qualidade de outras vacinas conjugadas contra bactérias encapsuladas de interesse epidemiológico no país. / Neisseria meningitidisgroup C is an encapsulated bacterium that causes several diseases and is associated with high mortality rates becoming a serious public health problem. Bio-Manguinhos is developing a conjugate vaccine constituted by covalent attachment of capsular polysaccharide to tetanus toxoid, which iscurrently being evaluated in Phase II clinical studies in children between 1-9 years. Free components quantification is a vaccine process control assay and intended to prevent exacerbated adverse reactions occurrence and/or vaccine immunogenicity reduction. The World Health Organization recommends 5% of free protein maximum level in the conjugate vaccine, but does not set a limit for the free polysaccharide contents. Thus, the aim of this study was to develop and validate quality control methods appropriate to separate and quantify free components present in the conjugate vaccine against N. meningitidisgroup C, using capillary electrophoresis (CE) technique. For free protein separation, free capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) were compared and different sample migration conditions were studied by varying parameters such as pH, temperature, voltage, buffer concentration, cyclodextrin and surfactant sodium dodecyl sulfate (SDS). The results showed that the best separation was obtained by MEKC using sodium tetraborate buffer (TBNA) 150 mM, 25 mM SDS, 60°C, 30 kV and pH 9.3. H owever, the CE did not induce a complete separation of the components suggesting that other techniques should be necessary. On the other hand, native and activated protein separation was possible using CZE, demonstrating the necessity of optimize protein activation reaction in order to increase the conjugation reaction yield. The total free sugar conjugate was completely separated from the conjugate by CZE using 50 mM TBNA buffer, 40°C, 30 kV and pH 10. In these conditions it was possible to determine the free polysaccharide content and validate the proposed method, which was linear in 0.047 to 0.164 mg/mL range, showed a matrix effect, 0.0154 mg/mL of detection limit and 0.0454 mg/mL ofquantification limit.After the validation steps, some conjugate batches were quantified and high levels of free sugar were observed in batches storaged at 4°C for long periods. On the other hand a conjugate batch recently produced was evaluated and showed19.08% of free polysaccharide. In order to estimate the maximum storage time a conjugate batch was analyzed30, 60 and 90 days after the production steps. The values found up to 60 days were not significantly different from that one determined at the initial time. However, with 90 days of storage there was a change in the conjugate profile that impaired its quantification. The methodology developed and validated will be used to evaluate the conjugate batch that will be submitted to Phase III clinical studies and in the routine quality controlof the conjugate vaccine. Moreover, the acquiredknowledge could be used in quality control of other conjugate vaccines against encapsulated bacteria of epidemiological importancein the country.

Neisseria gonorrhoeae (Ng) encounters different microenvironments during its life-cycle. Some of these niches have different concentrations of oxygen, which influences the rate of Ng growth; as well as iron, an element essential for Ng survival. Differential expression of several proteins allows the bacteria to adapt to the diverse conditions it comes encounters. One protein affected by environmental changes during Ng growth is Gcp, a tRNA-modification enzyme essential for protein synthesis. To study the regulation of expression of Gcp, we first analyzed the sequence of its ORF, gcp. Orthologs of this gene are found in all kingdoms of life. In silico analysis shows that among Neisseria species, gcp ranges in homology from 76% to 99%, at the nucleotide level. Reverse transcription PCR indicates that gcp is expressed as part of an operon, together with three cytochrome-associated genes cyc4, resB and resC. Rapid amplification of complementary DNA ends determined the start of transcription of cyc4 (and possibly of the cyc4-gcp operon) at 95 nucleotides from the gene start codon. Transcriptional fusions determined that the promoter region upstream of cyc4 is the strongest promoter in the operon. However, the region directly upstream of gcp also has low level of promoter activity, suggesting that the gene may be expressed from two different promoters. Semi-quantitative determination of the concentration of gcp mRNA indicates that the transcription of the gene is significantly repressed when Ng is grown under low iron or low oxygen conditions. Analysis of an fnr mutant, grown under the same conditions as its parental wild type, indicates that the FNR transcriptional regulator is involved in the repression of gcp in low iron or low oxygen conditions. Contrary to expectation, the cyc4 promoter is upregulated when Ng is grown under low oxygen or low iron conditions. However, these results cannot be compared to the original promoter strength. Determination of which was performed on bacteria grown in liquid medium. Coregulation of gcp with cytochrome genes can guarantee low levels of protein synthesis when Ng encounters adverse microenvironments and needs its energy redirected to the expression of genes that would allow it to survive.

In the search for a vaccine providing comprehensive protection against meningococcal disease, one vaccine currently under development contains the immunogenic proteins PorA and FetA in meningococcal outer membrane vesicles (OMVs). To achieve high levels of coverage against disease-causing isolates, the antigenic variability of these proteins could be overcome using knowledge of meningococcal epidemiology and population structure. In this study, the possible implications of variable expression levels of PorA and FetA on vaccine efficacy were investigated. Producing OMVs containing consistent amounts of FetA is difficult due to iron-repressed expression; therefore, meningococcal strains were constructed which constitutively expressed FetA at increased levels for OMV vaccine production and analysis. In mice, OMVs from modified strains induced antibodies against both PorA and FetA. These antibodies acted synergistically in a serum bactericidal assay; however, antibodies against FetA were weakly bactericidal alone. The potential to increase levels of PorA- and FetA-specific bactericidal antibodies with a prime-boost strategy, using OMV and protein inoculums, was also tested. While successful for a weakly-immunogenic PorA variant, a similar strategy did not increase bactericidal activity against FetA. Although antibodies against FetA can be induced following OMV immunisation, sufficient antigen expression in target bacteria is also required for bactericidal killing; therefore, the variability and regulation of porA and fetA transcription was investigated in a range of isolates. Despite differences in regulation among clonal complexes, variable expression is unlikely to be an issue for vaccine coverage. In particular, regulation of fetA by iron is reduced in many isolates due to a deletion in the sequence bound by the regulatory protein, Fur. Therefore, a vaccine targeting PorA and FetA may provide high levels of protection against meningococcal disease; however, an alternative formulation or immunisation strategy is required to improve coverage against FetA.

Bacteria of the Neisseria are predominately commensal, though N. gonorrhoeae and N. meningitidis are capable of causing disease. Both of these species often asymptomatically colonize humans, a trait reminiscent of their commensal cousins. The factors that shift the balance between asymptomatic carriage and disease are unknown. Pathogenic Neisseria use retractile surface structures called Type IV pili to coordinate community behavior and to initiate and sustain infection. Previously, the contributions of pilus retraction have been studied by deleting the pilus retraction motor, PilT. Recent findings suggest the speed and force exerted by pilus retraction is responsive to environmental cues. By examining several PilT mutants that maintain the ability to retract pili, I show retraction, per se, is not required for N. gonorrhoeae social interactions with bacteria or with human cells. Furthermore, Type IV pilus retraction by the commensal N. elongata affects the host cell differently than retraction by N. gonorrhoeae. These observations collectively suggest pilus retraction properties shape the host cell response to Neisseria colonization and could tip the balance of asymptomatic colonization to symptomatic disease.

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.

Neisseria gonorrhoeae is an obligate human pathogen that requires iron for its survival within the host. N. gonorrhoeae expresses high-affinity iron acquisition systems to acquire iron from host iron binding proteins. The gonococcal transferrin-iron uptake system is composed of two transferrin binding proteins, TbpA and TbpB. TbpA is a TonB-dependent, outer membrane transporter, while TbpB is a surface-exposed lipoprotein. Unlike TbpA, TbpB is not required for transferrin utilization, but makes the process more efficient. The precise mechanism by which TbpA and TbpB function to mediate transferrin-iron uptake has not been fully characterized. However, the mechanism of iron acquisition from transferrin is distinct from characterized TonB-dependent ferric-siderophore uptake systems. The transferrin-iron uptake system is unique in two ways: the involvement of the TbpB lipoprotein component and the process of iron acquisition and internalization. Unlike siderophore transporters, the transferrin-iron uptake system requires the removal of iron from transferrin for its subsequent internalization. Based on analogy with characterized TonB-dependent transporters, TbpA is proposed to consist of two distinct domains: a b-barrel and plug domain. Previous studies suggest that the plug domain has a specific role in iron internalization and this study addresses the role of the plug domain in transferrin-iron acquisition. It is thought that the TbpA plug domain facilitates iron removal from transferrin and subsequent iron binding and transport. To analyze this, iron binding by the TbpA plug domain was performed and site-directed substitution mutagenesis of putative iron-coordinating residues was carried out. From these analyses, it can be concluded that the plug domain binds iron and likely plays an active role in the process of iron internalization. Mutagenesis revealed specific residues of the plug domain critical for transferrin-iron uptake, but defects imparted by these mutations were compensated for by TbpB. Thus, this study also attempts to characterize the compensatory function provided by TbpB. Through mutagenesis, critical domains involved in the efficiency of transferrin-iron acquisition were identified. One additional study describes and characterizes a novel mechanism of TonB-independent transferrin-iron acquisition. Overall, these studies further elucidate mechanisms utilized by Neisseria gonorrhoeae in the process of iron acquisition from human transferrin.

Iron, an essential nutrient for most microorganisms, is sequestered in the host by iron-binding proteins, such as lactoferrin and transferrin. Neisseria gonorrhoeae utilizes transferrin as an iron source and its iron acquisition system is composed of two transferrin binding proteins: TbpA and TbpB. TbpA is a TonB-dependent, outer membrane transporter and TbpB is a bilobed, surface exposed lipoprotein. TbpB can distinguish between apo- and holo-transferrin which is involved in increasing the efficiency of iron uptake through the Tbps. It is anchored in the outer leaflet of the outer membrane by its lipid moiety. We aimed to identify the mechanism of TbpB export to the cell surface. No conclusions could be made from our results but we identified a protein that could potentially be involved in lipoprotein transport. TbpB is a bilobed protein with controversy over which lobe is involved in transferrin binding. In this study, we constructed a C-lobe deletion of TbpB to determine the role of the C-lobe in TbpB function. Results presented here showed deletion of the C-lobe caused degradation of TbpB and the minimal protein expressed was unable to bind transferrin both in vitro and in vivo. We were also able to demonstrate the TbpB C-lobe deletion is able to support limited transferrin-mediated growth, indicating some function of TbpB is retained. These results confirmed that both lobes are necessary for wild-type function of TbpB.

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.

RTX (Repeat in ToXin) superfamily consists of many proteins divided into several groups according to their different functions and characteristics: toxins, metalloproteases, lipases, proteins of the S-layer, bacteriocins and proteins with unknown function. However, all of them can be characterized by the following features: i) they contain tandemly repeated (6-50) nonapeptide glycine-rich calcium-binding consensus sequences GGXGXDX[L/I/V/W/Y/F]X (where X is any amino acid residue) in the C-terminal part of the protein. The presence of these repeats is a sine qua non condition for RTX protein family membership; ii) secretion from the cell occurs without a periplasmic intermediate by a mechanism which involves recognition of a signal sequence at the C-terminus of the protein by membrane-associated proteins that export the toxin across a channel spanning the entire bacterial envelope directly to the outside of the cell (Type I Secretion System); iii) the genes for protein synthesis, activation and secretion are mostly grouped together on the chromosome and form rtx operons. RTX toxins are the largest protein group of the RTX family. To this group belong mostly the proteins with molecular weight ranging from 100 to 200 kDa, with posttranslational fatty acid acylation mediated by a specific activating...

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.