Diss. (sammanfattning) Uppsala : Univ., 2003. / Härtill 3 uppsatser.

Diss. (sammanfattning) Stockholm : Univ., 2005. / Härtill 4 uppsatser.

Segundos mensageiros nucleotídicos são amplamente utilizados por bactérias para se adaptar às mudanças ambientais e fisiológicas. Neste cenário destaca-se o c-di-GMP, um segundo mensageiro praticamente universal em bactérias responsável por controlar a transição do estilo de vida bacteriano. Em geral, altos níveis celulares de c-di-GMP promovem um estado séssil, de formação de biofilme, enquanto baixos níveis induzem a motilidade. Xanthomonas citri subsp. citri (Xac), um fitopatógeno de grande importância econômica no Brasil, possui uma complexa regulação da sinalização de c-di-GMP, possuindo mais de 30 proteínas envolvidas na síntese, na degradação e na detecção deste segundo mensageiro. Dentre essas proteínas, destacam-se as que possuem os domínios de síntese e degradação presentes na mesma cadeia polipetídica, os domínios GGDEF e EAL respectivamente. A análise das estruturas primárias das 11 proteínas GGDEF-EAL codificadas pelo genoma de Xac revelou que a maior parte delas (6) provavelmente possui o domínio GGDEF inativo, enquanto o EAL é ativo. Três possivelmente possuem ambos os domínios ativos enquanto outras duas possuem ambos os domínios inativos. O nocaute do gene xac2382 que codifica uma dessas proteínas (que possui um domínio periplasmático seguido dos domínios citoplasmáticos HAMP-GGDEF-EAL), demonstra um aumento de motilidade e uma diminuição na formação de biofilme. Construções de fragmentos da proteína revelaram que XAC2382 necessita pelo menos dos domínios HAMP-GGDEF para complementar a cepa nocaute e que a atividade de diguanilato ciclase é essencial para isto. O domínio periplasmático de XAC2382 se mostrou interagir com XAC2383, uma proteína codificada por um gene presente no mesmo cluster do gene de XAC2382, e essa interação parece importante para o controle da motilidade de Xac. A estrutura de XAC2383 foi resolvida por cristalografia de raios X na qual foi revelada uma topologia típica de proteínas da família das periplasmic binding proteins (PBPs) possuindo ainda uma cavidade carregada positivamente contendo um motivo Ser-Thr-Ser (amnioácidos 152-154) importante para a ligação de compostos com grupos fosfatos ou fosfonatos. A mutação sítio dirigida nesse motivo aboliu os efeitos na motilidade dependentes dessa proteína. Esses resultados sugerem que XAC2383 é um sensor periplasmático de um composto eletronegativo e esta proteína interage com XAC2382 regulando a motilidade bacteriana. XAC0495, uma proteína com ambos os domínios GGDEF-EAL provavelmente inativos, pode fazer parte de um sistema de dois componentes com a histidina quinase XAC0494. XAC0495 se comporta como um monômero em solução e possui um formato alongado, como revelado por experimentos de SAXS. / Nucleotide based second messengers are widely used by bacteria in signaling pathways that mediate adaptations to environmental and physiological changes. c-di-GMP is a nucleotide second messenger ubiquitous in Gram-negative bacteria, where it plays a role in many important behaviors that define bacterial lifestyle. In general, high cellular levels of c-di-GMP promote biofilm formation, while low levels induce bacterial motility. Xanthomonas citri subsp. Citri (Xac), a pathogen of great economic importance in Brazil, has a complex repertoire of c-di-GMP signaling molecules, with more than 30 genes coding for proteins involved in the synthesis, degradation and detection of this second messenger. Among these proteins, many have both GGDEF and EAL domains (often associated with c-di-GMP synthesis and degradation, respectively) present in the same polypeptide chain. Analysis of the primary structure of 11 GGDEF-EAL proteins coded by the Xac genome revealed that six most likely possess an inactive GGDEF domain plus an active EALdomain. Another three proteins have both domains active while the other two have both domains inactive. The knockout of the xac2382 gene, coding for a protein which contains a periplasmic domain followed by cytoplasmic HAMP, GGDEF (active) and EAL (active) domains, shows an increase in motility and a decrease in biofilm formation. Constructions containing fragments of this protein revealed that constructs containing at least the HAMP and GGDEF domains are able to complement the knockout strain and that diguanilate cyclase activity is essential for this. The XAC2382 periplasmic domain was shown to interact with a protein encoded by a gene situated in the same cluster, XAC2383, and that this interaction seems crucial for the control of Xac motility. The structure of XAC2383 was solved by X-ray crystallography and was shown to adopt a topology typical of the periplasmic binding proteins (PBP) family. The protein possesses a positively charged groove that contains a Ser-Thr-Ser motif (152STS154) important for the binding of compounds with phosphate or phosphonate groups. Site-directed mutagenesis of this motif abolished the effects on motility caused by this protein. These results suggest that XAC2383 is a periplasmic protein responsible for sensing a compound with electronegative characteristics and which interacts with XAC2382, thereby regulating the bacterial motility. Another protein, XAC0495 (with both GGDEF-EAL domains probably inactive) may be part of a two-component system with the histidine kinase XAC0494. Small-angle X-ray scattering (SAXS) experiments reveal that XAC0495 exists as an elongated monomer in solution.

Segundos mensageiros nucleotídicos são amplamente utilizados por bactérias para se adaptar às mudanças ambientais e fisiológicas. Neste cenário destaca-se o c-di-GMP, um segundo mensageiro praticamente universal em bactérias responsável por controlar a transição do estilo de vida bacteriano. Em geral, altos níveis celulares de c-di-GMP promovem um estado séssil, de formação de biofilme, enquanto baixos níveis induzem a motilidade. Xanthomonas citri subsp. citri (Xac), um fitopatógeno de grande importância econômica no Brasil, possui uma complexa regulação da sinalização de c-di-GMP, possuindo mais de 30 proteínas envolvidas na síntese, na degradação e na detecção deste segundo mensageiro. Dentre essas proteínas, destacam-se as que possuem os domínios de síntese e degradação presentes na mesma cadeia polipetídica, os domínios GGDEF e EAL respectivamente. A análise das estruturas primárias das 11 proteínas GGDEF-EAL codificadas pelo genoma de Xac revelou que a maior parte delas (6) provavelmente possui o domínio GGDEF inativo, enquanto o EAL é ativo. Três possivelmente possuem ambos os domínios ativos enquanto outras duas possuem ambos os domínios inativos. O nocaute do gene xac2382 que codifica uma dessas proteínas (que possui um domínio periplasmático seguido dos domínios citoplasmáticos HAMP-GGDEF-EAL), demonstra um aumento de motilidade e uma diminuição na formação de biofilme. Construções de fragmentos da proteína revelaram que XAC2382 necessita pelo menos dos domínios HAMP-GGDEF para complementar a cepa nocaute e que a atividade de diguanilato ciclase é essencial para isto. O domínio periplasmático de XAC2382 se mostrou interagir com XAC2383, uma proteína codificada por um gene presente no mesmo cluster do gene de XAC2382, e essa interação parece importante para o controle da motilidade de Xac. A estrutura de XAC2383 foi resolvida por cristalografia de raios X na qual foi revelada uma topologia típica de proteínas da família das periplasmic binding proteins (PBPs) possuindo ainda uma cavidade carregada positivamente contendo um motivo Ser-Thr-Ser (amnioácidos 152-154) importante para a ligação de compostos com grupos fosfatos ou fosfonatos. A mutação sítio dirigida nesse motivo aboliu os efeitos na motilidade dependentes dessa proteína. Esses resultados sugerem que XAC2383 é um sensor periplasmático de um composto eletronegativo e esta proteína interage com XAC2382 regulando a motilidade bacteriana. XAC0495, uma proteína com ambos os domínios GGDEF-EAL provavelmente inativos, pode fazer parte de um sistema de dois componentes com a histidina quinase XAC0494. XAC0495 se comporta como um monômero em solução e possui um formato alongado, como revelado por experimentos de SAXS. / Nucleotide based second messengers are widely used by bacteria in signaling pathways that mediate adaptations to environmental and physiological changes. c-di-GMP is a nucleotide second messenger ubiquitous in Gram-negative bacteria, where it plays a role in many important behaviors that define bacterial lifestyle. In general, high cellular levels of c-di-GMP promote biofilm formation, while low levels induce bacterial motility. Xanthomonas citri subsp. Citri (Xac), a pathogen of great economic importance in Brazil, has a complex repertoire of c-di-GMP signaling molecules, with more than 30 genes coding for proteins involved in the synthesis, degradation and detection of this second messenger. Among these proteins, many have both GGDEF and EAL domains (often associated with c-di-GMP synthesis and degradation, respectively) present in the same polypeptide chain. Analysis of the primary structure of 11 GGDEF-EAL proteins coded by the Xac genome revealed that six most likely possess an inactive GGDEF domain plus an active EALdomain. Another three proteins have both domains active while the other two have both domains inactive. The knockout of the xac2382 gene, coding for a protein which contains a periplasmic domain followed by cytoplasmic HAMP, GGDEF (active) and EAL (active) domains, shows an increase in motility and a decrease in biofilm formation. Constructions containing fragments of this protein revealed that constructs containing at least the HAMP and GGDEF domains are able to complement the knockout strain and that diguanilate cyclase activity is essential for this. The XAC2382 periplasmic domain was shown to interact with a protein encoded by a gene situated in the same cluster, XAC2383, and that this interaction seems crucial for the control of Xac motility. The structure of XAC2383 was solved by X-ray crystallography and was shown to adopt a topology typical of the periplasmic binding proteins (PBP) family. The protein possesses a positively charged groove that contains a Ser-Thr-Ser motif (152STS154) important for the binding of compounds with phosphate or phosphonate groups. Site-directed mutagenesis of this motif abolished the effects on motility caused by this protein. These results suggest that XAC2383 is a periplasmic protein responsible for sensing a compound with electronegative characteristics and which interacts with XAC2382, thereby regulating the bacterial motility. Another protein, XAC0495 (with both GGDEF-EAL domains probably inactive) may be part of a two-component system with the histidine kinase XAC0494. Small-angle X-ray scattering (SAXS) experiments reveal that XAC0495 exists as an elongated monomer in solution.

L’agent pathogène Agrobacterium tumefaciens induit, chez les plantes, le développement de tumeurs dans lesquelles il prolifère, en intégrant un fragment de son plasmide Ti de virulence dans le génome de son hôte. Les tissus transformés synthétisent des composés originaux, appelés opines, qui sont utilisés comme nutriments spécifiques par la bactérie. Une vingtaine d’opines sont connues à ce jour, et chacune d’elle peut être métabolisée par des souches d’Agrobacterium tumefaciens possédant les gènes de transport et de catabolisme qui lui sont associés, ce qui apparait comme un avantage compétitif dans la colonisation de la tumeur. La présence de ces gènes dépend du type de plasmide Ti que la souche pathogène possède.Agrobacterium tumefaciens B6 possède un pTi de type octopine, qui porte les gènes de métabolisme des mannityl-opines, qui sont la mannopine, l’acide mannopinique, l’agropine et l’acide agropinique. La mannopine et l’acide mannopinique sont synthétisés par la même enzyme, et ont pour précurseurs respectivement la désoxy-fructosyl-glutamine (DFG) et le désoxy-fructosyl-glutamate (DFGA), tous deux opines de la famille de la chrysopine. La DFG est aussi un composé d’Amadori répandu et assimilable par de nombreux organismes. La mannopine sert de précurseur pour la synthèse de l’agropine. Enfin, la mannopine, l’acide mannopinique et l’agropine peuvent toutes trois se lactamiser spontanément en acide agropinique.Malgré la similarité chimique de ces quatre opines, chacune est transportée par une protéine périplasmique de fixation (PBP) associée à un transporteur ATP-binding Cassette (ABC) différent. La PBP sélectionne et fixe une opine pour l’apporter au transporteur ABC, qui permet le passage de l’opine dans le cytoplasme grâce à l’hydrolyse de deux molécules d’ATP. La spécificité du transporteur entier est déterminée par la PBP.Des études génétiques chez des souches possédant un pTi de type octopine ont montré que le système PBP-transporteur ABC AgaABCD est spécifique de l’acide agropinique, AgtABCD spécifique de l’agropine, MoaABCD spécifique de l’acide mannopinique et que MotABCD transporte la mannopine et également l’acide mannopinique. Chez la souche C58, qui ne possède pas un pTi de type octopine, le système de transport SocAB, codé par des gènes situés sur le plasmide cryptique At, transporte la DFG comme nutriment, et semble aussi capable d’importer la mannopine.Mon travail de thèse a permis, dans un premier temps, de caractériser les fortes affinités et la spécificité des PBP AgaA et AgtB pour l’acide agropinique, de la PBP MoaA pour l’acide mannopinique et de la PBP SocA pour la DFG, mais aussi la non spécificité de MotA pour la mannopine, l’acide mannopinique et la DFG, ce qui remet en question les affinités précédemment décrites pour AgtB et SocA. Dans un deuxième temps, ce travail a apporté les bases moléculaires et structurales des complexes PBP-mannityl-opines, complexes jamais caractérisés auparavant. Enfin, dans un troisième temps, la structure de la PBP AttC chez la souche C58, annotée comme mannopine-like, a été déterminée, et les expériences d’interaction ont montré qu’elle n’interagit avec aucune mannityl-opine, ce qui conduit à une révision de son annotation.Mes travaux apportent un éclairage nouveau sur l’import des mannityl-opines chez Agrobacterium tumefaciens. Le fait qu’aucun des transporteurs étudié ne permette l’import de l’agropine laisse penser qu’il existe une autre PBP ou un autre système de transport encore inconnu assurant cette fonction, ouvrant la voie vers de nouvelles études sur les pTi de type octopine et agropine. / Agrobacterium tumefaciens pathogenic agent confers the development of tumors in plants, in which it proliferates, integrating a fragment of its virulence Ti plasmid into its host genome. Transformed tissues synthesize original compounds, called opines, used as specific nutrients by the bacterium. More than twenty opines are known so far, and each one of them can be metabolized by A. tumefaciens strains possessing its associated transport and catabolism genes, which appears as a competitive advantage in the tumor colonization. The presence of these genes relies on the Ti plasmid type a pathogenic strain possesses. A. tumefaciens B6 possesses an octopine-type pTi, which harbors the metabolism genes of the mannityl-opines, which are mannopine, mannopinic acid, agropine and agropinic acid. Mannopine and mannopinic acid are synthesized by the same enzyme, and their precursors are deoxy-fructosyl-glutamine (DFG) and deoxy-fructosyl-glutamate (DFGA) respectively, both opines of the chrysopine family. DFG is also a wide-spread Amadori compound which can be uptaken by numerous organisms. Mannopine is a precursor for agropine synthesis. Finally, mannopine, mannopinic acid and agropine can spontaneously lactamize into agropinic acid.Despite the chemical similarity of these four opines, each one is transported by a different periplasmic binding protein (PBP) associated with an ATP-binding cassette (ABC) transporter. The PBP selects and binds one opine to bring it to the ABC transporter, which allows the passage of the opine to the cytoplasm due to two ATP molecules hydrolysis. The whole transporter specificity is determined by the PBP.Genetic studies in strains possessing an octopine-type pTi showed that AgaABCD PBP-ABC transporter system is specific to agropinic acid, AgtABCD to agropine, MoaABCD to mannopinic acid and that MotABCD transports mannopine and also mannopinic acid. In C58 strain, which do not possess an octopine-type pTi, SocAB transport system, coded by genes located on the cryptic pAt plasmid, allows the transport of DFG as a nutrient, and seems able to import mannopine too.My thesis work allowed, first, to characterize the strong affinities and the specificity of PBPs AgaA and AgtB to agropinic acid, PBP MoaA to mannopinic acid and PBP SocA to DFG, and also MotA unspecificity toward mannopine, mannopinique acid and DFG, which leads to a revision of the previously described affinities of AgtB and SocA. Secondly, this work brought molecular and structural basis of PBP-mannityl-opine complexes, never described before. Finally, the structure of PBP AttC, annotated as a mannopine binding-like protein in C58, was determined, and interactions experiments showed that it binds no mannityl-opines, leading to a revision of its annotation.My work sheds light on the mannityl-opines importation in Agrobacterium tumefaciens. The fact that none of the studied transport system allows agropine import lets think that there is another unknown PBP or another unknown whole transport system assuming this role, opening new ways to new studies about octopine- and agropine-type pTis.

Haemophilus influenzae is a pathogenic Gram-negative bacterium that colonizes the upper respiratory tract of humans and can cause otitis media, upper and lower respiratory infections, and meningitis. Factors important for H. influenzae to colonize humans and cause disease are not fully understood. Different bacterial pathogens are armed with virulence mechanisms unique to their specific strategies for interacting with their hosts. Many of the proteins mediating these interactions are secreted and contain disulfide bonds required for function or stability. I postulated that identifying the set of secreted proteins in H. influenzae that require periplasmic disulfide bonds would provide better understanding of this bacterium's pathogenic mechanisms. In this thesis, the periplasmic disulfide bond oxidoreductase protein, DsbA, was found to be essential for colonization and virulence of H. influenzae. Mutants of dsbA were also found to be sensitive to the bactericidal effects of serum. However, the DsbA-dependent proteins important for pathogenesis of this organism have not been previously identified. To find them, putative targets of the periplasmic disulfide bond pathway were identified and examined for factors which might be important for mediating critical virulence aspects. By doing so, novel virulence factors were discovered including those important for heme and zinc acquisition, as well as resistance to complement. Overall, the work presented here provides insight into requirements for H. influenzae to survive within various host environments.