OMP29 é uma das principais proteínas de membrana externa de Aggregatibacter actinomycetemcomitans (Aa) e está associada à invasão de célula epitelial gengival (CEG). Os objetivos deste estudo foram: analisar filogeneticamente omp29 e omp29 parálogo (omp29par), em cepas de Aa; determinar a interação de OMP29 com proteínas de matriz extracelular e o efeito da sua interação com CEG, pela avaliação da expressão gênica e produção de mediadores inflamatórios. Variações filogenéticas foram observadas para omp29 e omp29par, bem como para os seus promotores e estas relacionam-se com os sorotipos. A proteína recombinante OMP29his interagiu com fibronectina plasmática e celular (p<0,05), mas não com os domínios F30 e 45 ou com colágenos tipo I, III, IV e V, fibrinogênio, laminina e plasminogênio. A interação das mutantes de Aa deficientes em omp29 e/ou omp29par (obtidas pelo sistema LoxP/Cre) e OMP29his com CEG OBA-09 demonstrou que OMP29 regula positivamente il-18 e negativamente il-6r e il-8 (p<0,05). Os dados sugerem que OMP29 está envolvida na evasão do sistema imune. / OMP29 is one of the major outer membrane proteins of Aggregatibacter actinomycetemcomitans (Aa) and is associated with invasion into gingival epithelial cells (GEC). This study aimed to evaluate phylogenetically omp29 and omp29 paralogue (omp29par) in Aa strains; determine the interaction of OMP29 with extracellular matrix proteins and its effect on GEC by gene expression analysis and production of inflammatory mediators. Phylogenetic variations were observed for omp29 and omp29par as well as their promoters, and they were related to the serotypes. The recombinant protein OMP29his interacted with plasma and cellular fibronectin (p <0.05), but not to F30 and F45 domains, neither to collagens I, III, IV and V, fibrinogen, laminin and plasminogen. The interaction of Aa mutants defective in omp29 and/or omp29par (obtained by LoxP system/Cre) and OMP29his with CEG OBA-09 indicated that OMP29 regulates positively il-18 and negatively il-6r and il-8 (p <0.05). Data suggested that OMP29 is involved in bacterial evasion of the immune system.

Neisseria meningitidis B libera vesículas de membrana externa, conhecidas pela sigla OMV. Essas possuem os mesmos componentes da membrana externa da bactéria e podem ser utilizadas como antígenos em vacinas contra a meningite B. As vesículas devem, também, expressar proteínas da membrana externa (OMP) e proteínas reguladoras do íon ferro (IRP). O objetivo deste trabalho é estudar a cinética de crescimento bacteriano, consumo das fontes de carbono e nitrogênio - especialmente os limitantes de crescimento ? e produção de OMV visando melhorar a produção desse antígeno. Realizaram-se cultivos descontínuos em biorreator, com duração de 20 h, empregando meio de Catlin com limitação de ferro e modificações nas concentrações de lactato, aminoácidos e glicerol. As condições do cultivo foram: 4,2 L de meio, temperatura de 36°C, pressão de 0,5 atm, vazão de ar 1 L/min, agitação entre 250-850 rpm, controle de oxigênio dissolvido em 10% de saturação. Constatou-se que o lactato é a principal fonte de carbono limitante, embora somente se tem a hipótese de que o glicerol age como protetor mecânico. O ácido L-glutâmico é a principal fonte de nitrogênio consumida durante o cultivo. As OMV começaram a ser liberadas quantitativamente no início da fase estacionária de crescimento. Sendo que a melhor condição para a produção de OMV, valor 162,3 mg/L, é aquela em que as concentrações iniciais de lactato e aminoácidos foram duplicadas, 15,00 g/L e 2,93 g/L respectivamente. Através da análise do padrão eletroforético, confirmou-se a presença das principais proteínas de superfície, inclusive das IRPs. A integridade da OMV foi constatada por microscopia eletrônica. Assim, o antígeno obtido mostra-se passível de utilização na composição de vacina anti-meningocócica. / Neisseria meningitidis B liberates outer membrane vesicles known by the abbreviation OMV. These vesicles have the same components of the outer membrane of the bacteria and may be used as antigens in vaccines against meningitis B. The vesicles must also express outer membrane proteins (OMP) and iron regulated proteins (IRP). The aim of this paper is to study bacterial growth kinetics, carbon and nitrogen sources consumption ? specially those which limit growth ? and OMV production, seeking to improve the production of this antigen. Discontinuous bioreactor cultivations were carried out for a period of 20 hours in Catlin medium with iron restriction and modifications in lactate, amino acid, and glycerol concentrations. Cultivation conditions were: 4,2 L of medium, temperature at 36ºC, 0,5 atm, air flow rate of 1 L/min, agitation between 250-850 rpm, and dissolved oxygen control at 10% of saturation. It was verified that lactate is the main limiting carbon source, although there is just a hypothesis that glycerol acts as a mechanic protector. The L-glutamic acid is the main source of nitrogen consumed during the cultivation. The OMV started to be liberated quantitatively at the beginning of the stationary phase of growth. The best condition for production of OMV, value 162,3 mg/L, is that where the initial concentrations of lactate and amino acids were duplicated, 15,00 g/L and 2,93 g/L, respectively. Through an analysis of the electroforetic pattern, the presence of the main surface proteins was confirmed, including the IRPs. The integrity of the OMV was testified by electronic microscopy. So, the antigen thus obtained may be used in the antimeningococcal vaccine composition.

In Gram-negative bacteria, the three-layered cell envelope, including the cell wall, outer and inner membranes, is essential for cell survival in the changing, and often hostile environments. Conserved in all prokaryotes, the cell wall is incredibly thin, yet it functions to prevent osmotic lysis in diluted conditions. Based on observations obtained by genetic and chemical perturbations, time-lapse live cell imaging, quantitative imaging and statistical analysis, Part I of this dissertation explores the molecular and physical events leading to cell lysis induced by division-specific beta-lactams. We found that such lysis requires the complete assembly of all essential components of the cell division apparatus and the subsequent recruitment of hydrolytic amidases. We propose that division-specific beta-lactams lyze cells by inhibiting FtsI (PBP3) without perturbing the normal assembly of the cell division machinery and the consequent activation of cell wall hydrolases. On the other hand, we demonstrated that cell lysis by beta-lactams proceeds through four physical phases: elongation, bulge formation, bulge stagnation and lysis. Bulge formation dynamics is determined by the specific perturbation of the cell wall and outer membrane plays an independent role in stabilizing the bulge once it is formed. The stabilized bulge delays lysis, and allows escape and recovery upon drug removal. Asymmetrical in structure and unique to Gram-negative bacteria, outer membrane prevents the passage of many hydrophobic, toxic compounds. Together with inner membrane and the cell wall, three layers of the Gram-negative cell envelope must be well coordinated throughout the cell cycle to allow elongation and division. Part II of this dissertation explores the essentiality of the LPS layer, the outer leaflet of the outer membrane. Using a conditional mutant severely defective in LPS transport, we found that mutations in the initiation phase of fatty acid synthesis suppress cells defective in LPS transport. The suppressor cells are remarkably small with a 70% reduction in cell volume and a 50 % reduction in growth rate. They are also blind to nutrient excess with respect to cell size control. We propose a model where fatty acid synthesis regulates cell size in response to nutrient availability, thereby influencing growth rate. / Chemistry and Chemical Biology

Shigella is a genus of Gram-negative bacteria responsible for bacillary dysentery in humans. Shigella flexneri type 2a in particular is responsible for the majority of incidents in developing countries. The S. flexneri protease IcsP, is a member of the Omptin family of outer membrane (OM) proteases which cleaves IcsA, a polarly localised OM protein required for Shigella virulence. Mutations in icsP have been shown to effect the observed distribution of IcsA, however the significance of IcsP in Shigella virulence is incompletely understood. In this study, aspects of IcsP biology were investigated. S. flexneri 2457T and M90T icsP mutants were constructed to investigate the role of IcsP in Shigella intercellular spread, and it was found that icsP in both S. flexneri backgrounds did not appear to be essential for cell-tocell spread in human cervical cancer HeLa cells, but enhanced cell-to-cell spread in monkey kidney CV-1 cells (as determined by plaque assays). Complementation with icsP returned the mutant phenotype to wild-type. The results suggest IcsP does play a role in Shigella intercellular spread. The 2457T icsP mutant was subsequently complemented with an altered icsP gene encoding a haemagglutinin epitope tagged IcsP (IcsPHA) to determine the distribution of IcsP on the cell surface. In both S. flexneri and E. coli K-12 possessing smooth and rough lipopolysaccharide (LPS), the distribution of IcsPHA was found to be punctate across the cell surface. Deconvolution analysis revealed that IcsP distribution was punctate and banded in both LPS backgrounds. A smooth LPS E. coli K-12 yfdI mutant strain expressing IcsPHA was also constructed, and experiments involving treatment of this strain with bacteriophage Sf6 tail spike protein suggested that LPS O antigen chains masked IcsP in smooth LPS strains. During these studies, double-labelling of IcsPHA and LPS in a S. flexneri 5a M90T strain revealed a helical distribution of LPS in this strain. Overall, the results suggest IcsP has a punctate, banded distribution across the cell surface. The effect of virK and rmlD mutations on IcsP was then investigated by constructing a virK, rmlD and virK/rmlD double mutant in S. flexneri 2457T. Western immunoblotting showed no change in IcsP expression levels in either the virK, rmlD or virK/rmlD mutants compared to wild-type. Surprisingly, the virK mutant showed no change in IcsA expression levels by Western immunoblotting and plaque assays (using HeLa and CV-1 cells) suggested that virK was not essential for Shigella intercellular spread (contradicting the published data on this gene). No effect was also observed on IcsP expression level or on IcsP’s ability to cleave IcsA into culture supernatants. Finally alternative substrates for the protease activity of IcsP were investigated against known Omptin substrates (plasminogen, α2-antiplasmin, complement, protamine and colicins). However, IcsP appeared to have no effect on these substrates as determined by proteolytic cleavage assays and antimicrobial assay. Interestingly, Plg cleavage by rough LPS S. flexneri, and α2AP cleavage by both smooth and rough LPS S. flexneri, was observed. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1339487 / Thesis (Ph.D.) - University of Adelaide, School of Molecular and Biomedical Science, 2008

[Truncated abstract] Infectious and allergic diseases of the respiratory tract are major contributors to global mortality, morbidity and economic burden. Bacterial infections such as pneumonia and otitis media are important diseases, especially in children, while allergic diseases such as asthma and allergic rhinitis afflict up to 30% of the world's population. A confounding aspect of respiratory disease is the evidence of a complex relationship between respiratory allergy and respiratory infection, with infection suggested to both promote and prevent the pathogenesis of allergic disease. Additionally, allergy is a risk factor for bacterial infection such as otitis media, pneumonia and sinusitis, while respiratory infection can exacerbate allergic symptoms. Given the burden of bacterial respiratory disease and respiratory allergy, the development of preventative treatments for these diseases is needed and will benefit from clearer knowledge of the underlying immune mechanisms. This thesis aimed to to extend current knowledge by using Pasteurella pneumotropica, a similar bacteria to the human pathogen nontypeable Haemophilus influenzae (NTHi), to study respiratory infection and protective anti-outer membrane protein (OMP) immunity as well as the interaction of respiratory infection and allergic inflammation. Homologues of the important NTHi vaccine candidates P4, P6, P26 and D15 were found to be encoded by P. pneumotropica and a high level of amino acid sequence identity was noted between the different P. pneumotropica strains, as well as between other Pasteurellaceae members. ... In contrast, anti-P6his serum antibodies transferred to naïve mice did not confer protection. These results suggested that T-cell–mediated mechanisms were involved in P6his-mediated protection, and showed that the P. pneumotropcia model was useful for elucidating protective mechansims. The interaction of P. pneumotropica infection and papain-induced allergy was studied to investigate immune mechanisms underlying respiratory infection and allergy. Mice with ongoing allergic inflammation were intranasally challenged with bacteria and exhibited reduced pulmonary bacterial numbers, prolonged eosinophilia in the lungs and the induction of Th2 cytokines in the BALF, compared to nonallergic, infected mice. This suggested a protective role for allergic inflammation in this model. The effect of papaininduced inflammation on mice colonised by P. pneumotropica was also examined and allergic inflammation appeared to worsen infection in colonised mice. This suggested that allergic inflammation may also have a role in promoting infection in this model. In conclusion, this thesis explored mechanisms involved in vaccine-mediated immunity and the interaction of respiratory infection and allergy using a P. pneumotropica infection in its natural host. It was shown that intranasally administered recombinant P6 and P4 protected mice from lung infection, which justifies the inclusion of these OMPs as NTHi vaccine candidates. Additionally, it was demonstrated that the interaction of allergy and respiratory infection modulated immune responses. Overall, these results emphasize that a clearer understanding of the complex mechanisms underlying these interactions is required, and may be aided by the development of suitable animal models.

Shigella is a genus of Gram-negative bacteria responsible for bacillary dysentery in humans. Shigella flexneri type 2a in particular is responsible for the majority of incidents in developing countries. The S. flexneri protease IcsP, is a member of the Omptin family of outer membrane (OM) proteases which cleaves IcsA, a polarly localised OM protein required for Shigella virulence. Mutations in icsP have been shown to effect the observed distribution of IcsA, however the significance of IcsP in Shigella virulence is incompletely understood. In this study, aspects of IcsP biology were investigated. S. flexneri 2457T and M90T icsP mutants were constructed to investigate the role of IcsP in Shigella intercellular spread, and it was found that icsP in both S. flexneri backgrounds did not appear to be essential for cell-tocell spread in human cervical cancer HeLa cells, but enhanced cell-to-cell spread in monkey kidney CV-1 cells (as determined by plaque assays). Complementation with icsP returned the mutant phenotype to wild-type. The results suggest IcsP does play a role in Shigella intercellular spread. The 2457T icsP mutant was subsequently complemented with an altered icsP gene encoding a haemagglutinin epitope tagged IcsP (IcsPHA) to determine the distribution of IcsP on the cell surface. In both S. flexneri and E. coli K-12 possessing smooth and rough lipopolysaccharide (LPS), the distribution of IcsPHA was found to be punctate across the cell surface. Deconvolution analysis revealed that IcsP distribution was punctate and banded in both LPS backgrounds. A smooth LPS E. coli K-12 yfdI mutant strain expressing IcsPHA was also constructed, and experiments involving treatment of this strain with bacteriophage Sf6 tail spike protein suggested that LPS O antigen chains masked IcsP in smooth LPS strains. During these studies, double-labelling of IcsPHA and LPS in a S. flexneri 5a M90T strain revealed a helical distribution of LPS in this strain. Overall, the results suggest IcsP has a punctate, banded distribution across the cell surface. The effect of virK and rmlD mutations on IcsP was then investigated by constructing a virK, rmlD and virK/rmlD double mutant in S. flexneri 2457T. Western immunoblotting showed no change in IcsP expression levels in either the virK, rmlD or virK/rmlD mutants compared to wild-type. Surprisingly, the virK mutant showed no change in IcsA expression levels by Western immunoblotting and plaque assays (using HeLa and CV-1 cells) suggested that virK was not essential for Shigella intercellular spread (contradicting the published data on this gene). No effect was also observed on IcsP expression level or on IcsP’s ability to cleave IcsA into culture supernatants. Finally alternative substrates for the protease activity of IcsP were investigated against known Omptin substrates (plasminogen, α2-antiplasmin, complement, protamine and colicins). However, IcsP appeared to have no effect on these substrates as determined by proteolytic cleavage assays and antimicrobial assay. Interestingly, Plg cleavage by rough LPS S. flexneri, and α2AP cleavage by both smooth and rough LPS S. flexneri, was observed. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1339487 / Thesis (Ph.D.) - University of Adelaide, School of Molecular and Biomedical Science, 2008

Shigella is a genus of Gram-negative bacteria responsible for bacillary dysentery in humans. Shigella flexneri type 2a in particular is responsible for the majority of incidents in developing countries. The S. flexneri protease IcsP, is a member of the Omptin family of outer membrane (OM) proteases which cleaves IcsA, a polarly localised OM protein required for Shigella virulence. Mutations in icsP have been shown to effect the observed distribution of IcsA, however the significance of IcsP in Shigella virulence is incompletely understood. In this study, aspects of IcsP biology were investigated. S. flexneri 2457T and M90T icsP mutants were constructed to investigate the role of IcsP in Shigella intercellular spread, and it was found that icsP in both S. flexneri backgrounds did not appear to be essential for cell-tocell spread in human cervical cancer HeLa cells, but enhanced cell-to-cell spread in monkey kidney CV-1 cells (as determined by plaque assays). Complementation with icsP returned the mutant phenotype to wild-type. The results suggest IcsP does play a role in Shigella intercellular spread. The 2457T icsP mutant was subsequently complemented with an altered icsP gene encoding a haemagglutinin epitope tagged IcsP (IcsPHA) to determine the distribution of IcsP on the cell surface. In both S. flexneri and E. coli K-12 possessing smooth and rough lipopolysaccharide (LPS), the distribution of IcsPHA was found to be punctate across the cell surface. Deconvolution analysis revealed that IcsP distribution was punctate and banded in both LPS backgrounds. A smooth LPS E. coli K-12 yfdI mutant strain expressing IcsPHA was also constructed, and experiments involving treatment of this strain with bacteriophage Sf6 tail spike protein suggested that LPS O antigen chains masked IcsP in smooth LPS strains. During these studies, double-labelling of IcsPHA and LPS in a S. flexneri 5a M90T strain revealed a helical distribution of LPS in this strain. Overall, the results suggest IcsP has a punctate, banded distribution across the cell surface. The effect of virK and rmlD mutations on IcsP was then investigated by constructing a virK, rmlD and virK/rmlD double mutant in S. flexneri 2457T. Western immunoblotting showed no change in IcsP expression levels in either the virK, rmlD or virK/rmlD mutants compared to wild-type. Surprisingly, the virK mutant showed no change in IcsA expression levels by Western immunoblotting and plaque assays (using HeLa and CV-1 cells) suggested that virK was not essential for Shigella intercellular spread (contradicting the published data on this gene). No effect was also observed on IcsP expression level or on IcsP’s ability to cleave IcsA into culture supernatants. Finally alternative substrates for the protease activity of IcsP were investigated against known Omptin substrates (plasminogen, α2-antiplasmin, complement, protamine and colicins). However, IcsP appeared to have no effect on these substrates as determined by proteolytic cleavage assays and antimicrobial assay. Interestingly, Plg cleavage by rough LPS S. flexneri, and α2AP cleavage by both smooth and rough LPS S. flexneri, was observed. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1339487 / Thesis (Ph.D.) - University of Adelaide, School of Molecular and Biomedical Science, 2008

OMP29 é uma das principais proteínas de membrana externa de Aggregatibacter actinomycetemcomitans (Aa) e está associada à invasão de célula epitelial gengival (CEG). Os objetivos deste estudo foram: analisar filogeneticamente omp29 e omp29 parálogo (omp29par), em cepas de Aa; determinar a interação de OMP29 com proteínas de matriz extracelular e o efeito da sua interação com CEG, pela avaliação da expressão gênica e produção de mediadores inflamatórios. Variações filogenéticas foram observadas para omp29 e omp29par, bem como para os seus promotores e estas relacionam-se com os sorotipos. A proteína recombinante OMP29his interagiu com fibronectina plasmática e celular (p<0,05), mas não com os domínios F30 e 45 ou com colágenos tipo I, III, IV e V, fibrinogênio, laminina e plasminogênio. A interação das mutantes de Aa deficientes em omp29 e/ou omp29par (obtidas pelo sistema LoxP/Cre) e OMP29his com CEG OBA-09 demonstrou que OMP29 regula positivamente il-18 e negativamente il-6r e il-8 (p<0,05). Os dados sugerem que OMP29 está envolvida na evasão do sistema imune. / OMP29 is one of the major outer membrane proteins of Aggregatibacter actinomycetemcomitans (Aa) and is associated with invasion into gingival epithelial cells (GEC). This study aimed to evaluate phylogenetically omp29 and omp29 paralogue (omp29par) in Aa strains; determine the interaction of OMP29 with extracellular matrix proteins and its effect on GEC by gene expression analysis and production of inflammatory mediators. Phylogenetic variations were observed for omp29 and omp29par as well as their promoters, and they were related to the serotypes. The recombinant protein OMP29his interacted with plasma and cellular fibronectin (p <0.05), but not to F30 and F45 domains, neither to collagens I, III, IV and V, fibrinogen, laminin and plasminogen. The interaction of Aa mutants defective in omp29 and/or omp29par (obtained by LoxP system/Cre) and OMP29his with CEG OBA-09 indicated that OMP29 regulates positively il-18 and negatively il-6r and il-8 (p <0.05). Data suggested that OMP29 is involved in bacterial evasion of the immune system.

Orientador: Marcelo Lancellotti / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-23T11:46:02Z (GMT). No. of bitstreams: 1 Mattos_IvesBernardellide_M.pdf: 15060536 bytes, checksum: 32a633419d15c105fec735cb5753d5b2 (MD5) Previous issue date: 2012 / Resumo: O resumo poderá ser visualizado no texto completo da tese digital quando liberada / Abstract: The abstract is available with the full electronic document when available / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular

The pathogenic members of the genus Yersinia, Y. pseudotuberculosis, Y. pestis and Y. enterocolitica are transmitted from animals to man and may give rise to disease with a variety of symptoms. These bacteria possess related plasmids necessary for virulence. In this study, gene products encoded by the virulence plasmid have been identified and characterized. A temperature-inducible outer membrane protein YOP1, is encoded by the virulence plasmid. YOP1 is expressed by Y. pseudotuberculosis and Y. enterocolitica at 37°C. The genetic locale of trie structural gene for YOPl on the virulence plasmid was determined. A mutant that was unable to express this protein, remained fully virulent, showing that YOP1 is not a virulence determinant. Several other proteins encoded by the virulence plasmid are induced at 37°C in a medium lacking Ca2+. These proteins are not expressed at 26°C and expression is repressed by Ca2+-concentrations in excess of 2.5 mM. In Ca2+-deficient medium, the induced proteins can be found extracellu- larly as well as in the outer membrane. However, in the presence of Ca at 37°C they are only found in the outer membrane. The released proteins consist of eight polypeptides as revealed by two-dimensional electro­phoresis. These proteins, Y0P2a and 2b, YOP3, Y0P4a and 4b, the V-antigen and a small uncharacterized polypeptide, are expressed by all three pathogenic Yersinia species, both in vivo and in vitro. The Ca2+-controlled expression of the YOP proteins is regulated by genes in the Ca2+ -region, which are conserved in the three species. Mutations in this region repress the expression of the Ca2+-regulated YOPs. The genetic loci identified for five of these proteins revealed that only the structural gene of the Y0P4b protein is part of the Ca2+ -region. The other genes were found at separate locations outside this region. The structural genes for YOP4b, YOP3 and the V-antigen, together with the genes for two additional polypeptides, were localized to a common region conserved on the plasmids of the Yersinia species. The structural genes for Y0P2b (yopH) and Y0P5 (yopE) are located in different positions on the plasmid from Y. enterocolitica, compared to the other two species. This plasmid has Been rearranged so that these genes are located close to one another. The DNA sequence of the yopH gene shows that it is a singly transcrip­tional unit. Transcription of this gene is regulated by Ca2+-concentra­tion and by temperature. A mutant strain of Y. pseudo tuberculosis, de­leted for the yopH gene on the virulence plasmid, is avirulent In mice. Virulence is restored by trans-complementation with the cloned yopH gene. The mutant strain is also’ unable to inhibit phagocytosis of macrophages as compared to the wild-type strain. The trans-compleroented strain shows inhibition comparable to that of the wild-type. Therefore, the YOP2b protein is considered to be an essential virulence determinant. / digitalisering@umu.se