Bacillus subtilis é uma alternativa ao emprego de Escherichia coli para a produção de proteínas recombinantes. O principal entrave à utilização de B.subtilis para esse fim é a baixa disponibilidade de sistemas de expressão. Nesse trabalho, testamos diferentes plasmídeos e promotores com o objetivo de desenvolver sistemas de expressão heteróloga eficientes. Ao fim do trabalho, propomos dois novos sistemas de expressão baseados do arcabouço do plasmídeo pMTL500E e nos promotores dos genes cdd e gsiB, ambos de B.subtilis. Os dois plasmídeos construídos apresentam expressão constitutiva e demonstraram desempenho superior no tocante à produção de proteínas heterólogas quando comparados ao único sistema comercialmente disponível, conhecido como pHT01. Em uma segunda parte do trabalho, propomos a utilização de Listeria innocua como veículo de entrega para antígenos vacinais. Por meio de ensaios ex-vivo e in vivo, demonstramos que essa bactéria possui potencial promissor para aplicações vacinais, inclusive quando comparada ao bem estabelecido B.subtilis. / Bacillus subtilis is an alternative to the use of Escherichia coli for the production of recombinant proteins. The main bottleneck to the use of B. subtilis for this purpose is the low availability of expression systems. In this study, we evaluated different plasmids and promoters with the aim of developing efficient heterologous expression systems. At the end of this work, we propose two new expression systems based on plasmid pMTL500E and the promoters from cdd and gsiB genes, both of them from B.subtilis. The two plasmids constructed exhibit constitutive expression and demonstrated superior performance regarding the production of heterologous proteins compared to the unique commercially available system, which is known as pHT01. In a second part of the work, we propose the use of Listeria innocua as a delivery vehicle for vaccine antigens. After ex-vivo and in-vivo experiments, we demonstrated that this bacterium has a promising potential for vaccine applications, even when compared to well established B.subtilis.

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

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

Diss. (sammanfattning) Linköping : Linköpings universitet, 2010. / Härtill 4 uppsatser.