Haemophilus influenzae tipo b é uma bactéria Gram-negativa, patogênica causadora de meningites em crianças. A cápsula polissacarídica (PSb) é o principal fator de virulência e é usado como antígeno vacinal. O método clássico de purificação do PSb envolve várias etapas de precipitação com etanol, fenol e detergente catiônico (inflamável, corrosivo e tóxico), e etapas de ultracentrifugação. O objetivo deste estudo foi substituir total ou parcial as precipitações e/ou uso das centrífugas por cromatografia, digestão enzimática, microfiltração e ultrafiltração tangencial. As cromatografias de troca iônica e de filtração em gel não apresentaram boas purificações, entretanto a hidrofóbica pode eliminar as proteínas contaminantes. As precipitações com etanol foram necessárias para obter a pureza requerida. O etanol de alguma forma favoreceu a ação enzimática e facilitou a posterior ultrafiltração. A separação com etanol em fibra-oca de microfiltração tangencial mostrou melhores purificações do que a centrifugação, mas com uso repetido verificou-se redução na eficiência. / Haemophilus influenzae type b is Gram-negative pathogenic bacterium cause meningitis in children. The capsular polysaccharide (PSb) is the main virulence factor and it is used as vaccine antigen. The classical PSb purification process includes ethanol, phenol and cationic detergent precipitations (explosion prone, corrosive, toxic) and ultracentrifugation steps. The aim of this work was to replace total or partial ethanol precipitations steps and/or elimination of centrifugation by chromatography methods, enzymatic digestion and ultrafiltration (UF) or microfiltration. The results have showed that ion exchange chromatography and gel filtration did not result in good purification, however the hydrophobic can be used for proteins elimination. The ethanol precipitation steps are necessary to achieve the required purity of PSb. In some way ethanol contributed for enzymes action and further improvements in the UF. The ethanol separation with hollow fiber microfiltration exhibited better purification than centrifugation, but after some uses the efficiency has reduced.

Haemophilus influenzae b (Hib) é uma bactéria patogênica causadora de pneumonia e meningite. Sua cápsula polissacarídica (PRP) é considerada como principal fator de virulência e utilizada como antígeno vacinal. Hib é fastidioso e requer micronutrientes para seu desenvolvimento. A finalidade deste trabalho é estabelecer o meio quimicamente definido para desenvolvimento de Hib e produção de PRP. Inicialmente, definiu-se um meio a partir de dados da literatura. Este meio foi estudado através do delineamento de Plackett-Burman de 44 ensaios, obtendo-se valores máximos de DO540nm de 5,0 UA, e 227,7 mg/L de PRP. A análise estatística revelou que EDTA, NH4Cl, Cys e PVA podem ser removidos do meio sem impactar os parâmetros estudados e que Glm, Hipoxantina, Inosina, Tiamina, Hemina e Tween 80 apresentam efeito significativo positivo para produção de PRP. Analisando os meios estudados, foi possível verificar que a composição do E44 possibilitou produzir o PRP a US$ 16,50/g, sendo considerado o meio quimicamente definido estabelecido neste trabalho. / Haemophilus influenzae b (Hib) is a pathogenic bacterium that causes pneumonia and meningitis. Its capsular polysaccharide (PRP) is considered as a major virulence factor and used as vaccine antigen. Hib is fastidious and requires micronutrients for its development. The purpose of this study is to establish the chemically defined medium for Hib development and PRP production. Initially, a medium was defined based in the literature. This medium was studied by the Plackett-Burman design of 44 trials, achieving maximum values of DO540nm of 5.0 AU and 227.7 mg / L of PRP. Statistical analysis revealed that EDTA, NH4Cl, Cys and PVA can be removed from the medium without impacting the parameters studied and Glm, Hypoxanthine, Inosine, Thiamine, Tween 80 and Hemin exhibit significant positive effect on the PRP production. Analyzing the studied media, it was possible to verify that the composition of E44 enabled to produce PRP to $ 16.50/g, being considered the chemically defined medium established in this work.

Historically, the causative agent of infectious coryza has been identified as the NAD requiring bacterium Haemophilus paragallinarum and the implementation of an intensive vaccination program led to the effective control of this contagious upper respiratory infection. More recently, however, a decline in the protective capacity of a vaccine conditioned immune response was noted, with a number of contributing factors, including the emergence of a fast-growing NAD-independent bacterium, which has largely replaced the traditional NAD-dependent variety. As such, accurate, reproducible methods for determining and continually monitoring the type of infecting bacteria was necessitated. To address this need, strains of H. paragallinarum were evaluated according to both their phenotypic and their genotypic properties, in a combination serodiagnostic approach. A data bank of NAD-dependent H. paragallinarum reference strain and field isolate serovar-specific fingerprints was established on both a whole cell and outer membrane protein level. Visual comparative analysis of the qualitatively and quantitatively similar outer membrane protein patterns of all strains of NAD independency studied with the formulated data bank, indicate that the NAD-independent strains displayed profiles typical of serovar C-3. The outer membrane proteins have been identified as putative virulence determinants and, as such, were characterised according to their surface location, susceptibility to heat modification, functional role as endotoxins, sequence homology to structural membrane counterparts, and finally, their ability to induce an immune response. These studies represent novel efforts and form the foundation for identifying those antigens responsible for maintaining an infection in the host milieu. Ribotype analysis served as an adjunct to phenotypic observations, with the local NAD-independent field isolates being identified as serotype A. These contradictory outcomes call for the creation of a set of reference strains specific for NAD-independent isolates. The identification of restriction fragment length polymorphisms in the conserved 16S rRNA gene sequences indicate the potential application of this method for type assignment, requiring the recognition of a battery of versatile restriction enzymes to generate serovar-specific polymorphic profiles. The complexity of serotype allocation demands that a combination approach in which genotypic analyses complement phenotypic-based methods of haemagglutination inhibition and outer membrane protein profiling. The groundwork for implementation of such a system has been accomplished. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1998.

Haemophilus influenzae is a host adapted human mucosal pathogen involved in a variety of acute and chronic respiratory tract infections, including chronic obstructive pulmonary disease and asthma, all of which rely on its ability to efficiently establish continuing interactions with the host. Here we report the characterization of a novel molybdenum enzyme, TorZ/MtsZ that supports interactions of H. influenzae with host cells during growth in oxygen-limited environments. Strains lacking TorZ/MtsZ showed a reduced ability to survive in contact with epithelial cells as shown by immunofluorescence microscopy and adherence/invasion assays. This included a reduction in the ability of the strain to invade human epithelial cells, a trait that could be linked to the persistence of H. influenzae. The observation that in a murine model of H. influenzae infection, strains lacking TorZ/MtsZ were almost undetectable after 72 h of infection, while similar to 3.6 x 10(3) CFU/mL of the wild type strain were measured under the same conditions is consistent with this view. To understand how TorZ/MtsZ mediates this effect we purified and characterized the enzyme, and were able to show that it is an S- and N-oxide reductase with a stereospecificity for S-sulfoxides. The enzyme converts two physiologically relevant sulfoxides, biotin sulfoxide and methionine sulfoxide (MetSO), with the kinetic parameters suggesting that MetSO is the natural substrate of this enzyme. TorZ/MtsZ was unable to repair sulfoxides in oxidized Calmodulin, suggesting that a role in cell metabolism/energy generation and not protein repair is the key function of this enzyme. Phylogenetic analyses showed that H. influenzae TorZ/MtsZ is only distantly related to the Escherichia colt TorZ TMAO reductase, but instead is a representative of a new, previously uncharacterized Glade of molybdenum enzyme that is widely distributed within the Pasteurellaceae family of pathogenic bacteria. It is likely that MtsZ/TorZ has a similar role in supporting host/pathogen interactions in other members of the Pasteurellaceae, which includes both human and animal pathogens.

Acute respiratory tract infections are the most common cause o f illness and death in the pediatric population worldwide. It is estimated that 70 - 80% o f severe pneumonias in Africa are caused by S.pnewnoniae (the pneumococcus) followed by H. influenzae type b. Surveillance reveals that drug resistance is increasing worldwide, South Africa not being an exception. This has considerably complicated the management o f infections caused by both the pneumococcus and H. influenzae type b ( H ib ). It is widely accepted that colonization of the nasopharynx even briefly precedes middle ear infection and invasive pneumococcal disease. Early onset of colonization after birth has been associated with early onset o f middle ear infections. Furthermore, colonized children are able to transmit these organisms to other children. Carriage o f pneumococci commonly occurs in young children. The carriage of resistant pneumococci is usually limited to those serotypes carried in children. N ew conjugate vaccines may be able to reduce colonization o f these serotypes. This study was undertaken to determine the serotypes and susceptibility o f pneumococci and H. influenzae type b, and the proportion o f healthy children colonized at Zola Community Health Centre (ZCHC) in Soweto.

A Febre Purpúrica Brasileira - FPB- foi reconhecida como uma doença pediátrica fulminante, caracterizada por febre alta com rápida progressão para púrpura, choque e óbito. A grande maioria dos pacientes apresentaram conjuntivite prévia. O presente trabalho procura descrever os aspectos epidemiológicos desta doença, desde o surgimento dos primeiros casos, em 1984, em Promissão, Estado de São Paulo, Brasil. Foram registrados 277 casos distribuídos no Brasil em cinco Estados: São Paulo, Paraná, Mato Grosso, Mato Grosso do Sul e Minas Gerais. Fora do Brasil, dois casos foram relatados na Austrália. Cerca de 89% dos casos ocorreram em crianças de até cinco anos de idade. A letalidade foi de 38%. Estima-se o período de incubação médio de 15 dias. O quadro inicial se caracteriza por conjuntivite e febre. O agente etiológico é a bactéria Haemophilus influenzae biogrupo aegyptius, clone invasivo, tendo sido isolado de sangue, liquor, lesão hemorrágica de pele, secreção de conjuntiva e de orofaringe em 1986, de pacientes no Estado de São Paulo. Os estudos moleculares identificaram características diferentes das descritas até então para o Haemophilus aegyptius isolado de surtos de conjuntivite. Os principais surtos da doença ocorreram em Serrana, Valparaíso no Estado de São Paulo e Maracaju no Estado do Mato Grosso do Sul. Em algumas localidades com surtos de conjuntivite se observou a presença de moscas (Diptera). Acredita-se que estes insetos tenham participação na disseminação da doença. O diagnóstico precoce da FPB é um fator importante para redução da letalidade. / Brazilian Purpuric Fever- BPF- has been recognized as a fulminant pediatric disease characterized by fever with rapid progression to purpura, shock and death. The vast majority of BPF patients have previously presented conjunctivitis. The present work aims to describe epidemiological aspects of BPF since the appearance of the first cases, in 1984, in Promissão, State of São Paulo, Brazil. It has been reported 277 cases in Brazil, distributed by five states: São Paulo, Paraná, Mato Grosso, Mato Grosso do Sul and Minas Gerais. Outside Brazil, only two cases have been reported in Australia. About 89% from the total number of cases occurred in children aging five years old or less. The case fatality rate was 38%. The average incubation period is estimated as being 15 days. The initial clinical symptoms are conjunctivitis and fever. The etiologic agent of BPF is the bacteria Haemophilus influenzae biogroup aegyptius, invasive clone. It has been isolated from blood, cerebrospinal fluid, hemorrhagic skin lesion and conjunctival and orofarynx secretions in 1986, from São Paulo State patients. Molecular studies have identified different features from those described to H.aegyptius which have been isolated during conjunctivitis outbreaks since then. The most important BPF outbreaks occurred in Serrana and Valparaíso, State of São Paulo and in Maracaju, State of Mato Grosso do Sul. The presence of gnats (Diptera) has been observed in some of the places where conjunctivitis outbreaks have occurred. It is believed that these insects are associated with BPF transmission. Early diagnosis is an important factor in fatality reduction.

Nontypeable Haemophilus influenzae (NTHi), Streptococcus pneumoniae and Moraxella catarrhalis are common bacterial agents of otitis media which is a major cause of morbidity in young children. Mucosal immune responses are an integral part of the immune defense against middle ear infection and it is known that certain populations, including Australian Aboriginal children, are highly susceptible to disease. The current study focussed on the development of the mucosal immunity to the three bacterial pathogens in Aboriginal and non-Aboriginal children from birth to two years of age, living in the Kalgoorlie-Boulder region of Western Australia. Salivary and breast milk IgA levels were measured by the enzyme Linked immunosorbent assay. The measured IgA levels, combined with socio-economic, demographic and bacteriological data were analyzed statistically to determine the influential factors on the mucosal IgA response in these children over time. This study found that each antigen-specific IgA examined followed a distinct ontogeny pattern and IgA responses differed significantly according to age, indigenous status and feeding type. Indoors smoke exposure, maternal smoking, and sibling day care attendance had some impact on salivary IgA levels in the children. However, household crowding and the presence of older siblings had the most significant impact on salivary IgA levels for children of different age groups. These two factors were correlated to increased nasophayrngeal colonization by H. influenzae, S. pneumoniae and M. catarrhalis and colonization status was also found to influence salivary IgA levels in the children. No correlation between maternal breast milk IgA levels and child salivary IgA levels was observed. The results suggest that the degree of exposure to environmental factors rather than immunological deficit is responsible for the observed differences in salivary IgA responses between Aboriginal and non-Aboriginal children and modifying these factors could lead to a reduction in the burden of otitis media experienced by the children. Further studies correlating specific salivary IgA levels to diseases such as otitis media will reveal the role of specific salivary IgA responses in the prevention of infection by respiratory pathogens.

Nontypeable Haemophilus influenzas (NTHi) is recognised as a significant human pathogen causing mild to severe respiratory tract infections. At present, no vaccine is available for prevention of infection caused by this pathogen. Several outer membrane proteins (OMPs) of NTHi and its lipooligosaccharide have been investigated as possible vaccine antigens against NTHi infections. Previous investigations in our laboratory have shown that OMP26 from an NTHi 289 strain was able to significantly enhance pulmonary clearance of NTHi in a rat model in which animals were immunised via intestinal Peyer's patches and then boosted intratracheally (Kyd and Cripps, 1998; El- Adhami et al., 1999). In recent studies, the OMP26, when used as a parenteral immunogen, was also highly effective at inducing immune responses that led to significantly enhanced clearance of the chinchilla nasopharynx (Kyd et al., 2003). These studies indicate significant potential of the OMP26 as a candidate vaccine antigen and warrant further investigations for development of a vaccine against NTHi. This thesis focussed on the immunological and structural characterisation of the NTHi vaccine candidate, OMP26. Peptides of OMP26 were used as tools to localise the immunologically important regions of the OMP26. Two different E. coli expression systems, the GST gene fusion and the 6xHis tagged systems, were employed to construct the OMP26 peptides. It was found in this study that, despite efforts to optimise the system, the GST-fusion protein system failed to produce consistent results for the purification and storage of the OMP26 peptides. In contrast, the 6xHis tagged system exhibited more reliable outcomes in the production of the recombinant OMP26 peptides and the stability of the stored purified peptides. As such, the purified OMP26 peptides from the 6xHis tagged system were chosen to map major regions of immunological significance for the OMP26 protein. The regions of the OMP26 which are involved in the induction of the acquired immune responses have been identified in the present study. Based on the antigen specific lymphocyte proliferation assay, the dominant T cell epitopes for OMP26 were located between amino acid residues 95 and 197 (T3+T4 region). These identified T cell epitopes exhibited the capability of efficient T cell activation, suggesting that the epitopes within the T3+T4 region potentially had the highest affinity for binding to the MHC molecules than did any other OMP26 region. Using two different assay systems, ELISA and BIA, the predominant B cell epitopes of OMP26 were located between amino acid residues 45 and 145 (T2+T3 region). This region was also found to be immunodominant across all animal species tested, and with all immunisation regimens used. Flow cytometry analysis also revealed that these particular epitopes were expressed on the surface of NTHi cells. By integration of the data obtained from these current experimental studies and the computational analysis of the OMP26 sequence, two hypothetical models of the OMP26 were also proposed in this study. The significant outcomes obtained in this thesis provide a better understanding of the specificity of the host immune responses to the OMP26 protein These findings provide great benefit not only for the development of a future NTHi vaccine but for the development of the peptide-based immunodiagnostic reagents as well. These diagnostic reagents will be valuable, in particular, for the evaluation of efficacy of an NTHi vaccine in humans that may include OMP26 or specific conformational structures. Future studies are still required to further define the minimum epitope length required for the B and T cell responses identified in this study. The significance of these responses in immune protection against NTHi infection also requires further investigations. Human immune responses also need to be determined, but this can only be achieved following clinical trial studies.

Previous work carried out in our laboratory has identified that phase variation of type III R-M systems found in Haemophilus influenzae, Neisseria meningitidis and N. gonorrhoeae is reversible, and occurs at high frequency, as seen both through mod::lacZ fusions, and by measuring changes in repeat tract length. In addition, phase variation of the methyltransferases results in coordinated switching of expression of a distinct group of genes in each of the strains studied so far. WE have termed this phenomenon the PHASEVARION, for phase variable regulon, to identify the set of genes whose expression is affected by moe phase variation. Many of the genes found to be regulated by mod phase variation are known virulence factors and even include some genes investigated as candidates for vaccine development (Srikhanta et al., 2005 and 2009. The aims of this project was to further the investigation of how these R-M systems regulated the expression of genes which hitherto had not been predicted to phase vary. The first step in the process of investigating how phase variable R-M systems influence expression of unrelated genes is to identify the DNA sequences methylated by the methyltransferases of interest. As discussed in Chapter 3, elucidation of the ModA1 methylation target site was in part facilitated by predictions that the phase variable methyltransferase found in H. influenzae strain Rd methylated the same sequence as did HinfIII, isolated from H. influenzae strain Rf. This hypothesis was confirmed by methylation dependent inhibition of digestion, revealing that ModA1 methylates the second A in its recognition sequence, 5’-CGAAT-3’. Once confirmed, the genes found to be regulated by modA1 phase variation in the initial phasevarion study could be investigated for the presence of ModA1 methylation sites within their promoters or upstream of their transcriptional regulators. Two such methylation target sites were located just upstream of the dnaK ORF. Transcriptional start site analysis of the dnaK gene revealed three transcripiotnal start sites, one of which is unduced by heat shock. Exactly 10 nucleotides upstream of this heat shock induced transcriptional start site lies one of these ModA1 methylation target sequences. Ongoing invetigations are looking into the importance of this ModA1 site located within the dnaK promoter, and whether this is the site responsible for ModA1 dependent variations in dnaK expression. Although numerous methods were investigated for their potential to identify all sites methylated by the different modA alleles, the only method which resulted in identification of any methylation target sites was methylation dependent inhibition of restriction. This method allowed us to confirm the ModA1 recongition sequence, and to discover the methylation sequence, and adenine targeted by the modA13 allele, which is found in many clinically relevant N. gonorrhoeae strains. As will be discussed in Chapter 5, ModA13 dependent inhibition of restriction was first observed when the Neisserial plasmid pCmGFP was extracted from modA13 ON and modA13::kan cells, and further investigated and confirmed using a Southern blot approach to determine whether ModA13 dependent inhibtion could be detected as differential methylation of the chromosome. It was found that ModA13 recognised the sequence 5’-AGAAA-3’, with methylation occurring on the second last A. This sequence was mapped not only to the genes found to be regulated by modA13 phase variation, but also to the entire FA1090 chromosome, and this information will be used in future studies to investigate the direct molecular mechanisms by which modA13 phase variation results in subpopulations with different phenotypes in relation to antimicrobial resistance and biofilm/cell invasion.

PCR is a rapid, reproducible method for nucleic acid detection. However, this technology displays significant deficiencies when applied in clinical microbiology. This work’s aim was to improve current diagnostics and provide sensitive and quantitative real-time PCRs. Paper I describes the development of a sensitive and specific quantitative real-time PCR for the detection of Streptococcus pneumoniae, based on the Spn9802 DNA fragment. Applied to nasopharyngeal aspirates from 166 pneumonia patients, Spn9802 PCR had a sensitivity of 94% and a specificity of 98%. In Paper II the performance of a ply gene PCR for identification of pneumococcal lower respiratory tract infection (LRTI) was evaluated on bronchoalveloar lavage fluids. At the detection limit 103 genome copies/mL, 89% sensitivity but only 43% specificity was achieved. Paper III shows that S. pneumoniae DNA is detectable in plasma from acutely febrile patients. Sensitivities were low (26-42%) for detection of pneumococcal pneumonia, for bacteraemic pneumococcal pneumonia they were 60-70%. Paper IV describes evaluation of four PCR targets for Haemophilus influenzae detection. A real-time PCR based on the P6 gene was developed and applied to 166 CAP patients, using cut-off of 104 genome copies/mL the assay had a sensitivity of 97% and a specificity of 96%. In paper V, the two real-time PCRs presented in papers I and IV were combined with a PCR for detection of Neisseriae meningitidis. The analytical sensitivity of this multiplex real-time PCR was not affected by using a mixture of reagents and a combined DNA standard (S. pneumoniae/H. influenzae) in single tubes. Applied to 156 LRTI patients, this PCR had sensitivities over 90% for S. pneumoniae and H. influenzae, and specificities of 89% and 96%, respectively. In conclusion, real-time PCR assays are useful for the diagnosis of S. pneumoniae and H. influenzae. They enable detection after antibiotic installation, and quantification increases the etiological specificity of pneumonia.