O sistema de transporte de oligopeptídios (sistema Opp) está envolvido em diferentes aspectos da fisiologia bacteriana, incluindo nutrição, comunicação intercelular e fatores associados com a virulência. Estes transportadores ABC são formados por uma proteína de ligação a oligopeptídios, uma permease e um domínio de ligação ao ATP. As Aeromonas são bactérias Gram-negativas aquáticas ubíquas associadas com várias doenças em humanos, especialmente, gastrenterites. Atualmente, A. hydrophila, A. sobria e A. caviae são consideradas como patogenos emergentes pela OMS. Neste contexto, o objetivo do presente trabalho foi caracterizar o sistema de transporte de oligopeptídios em Aeromonas utilizando para tanto diversas ferramentas bioinformáticas e moleculares. Os resultados mostraram que, assim como em outras bactérias Gram-negativas, os genes opp de Aeromonas encontram-se organizados em um único operon policistrônico formado por cinco genes (oppA, oppB, oppC, oppD e oppF). O gene oppA e a proteína periplásmica de ligação a oligopeptídios correspondente (OppA) são altamente conservados, mesmo entre bactérias de famílias distintas. O modelo da proteína OppA de A. hydrophila mostrou a estrutura típica Venus flytrap , semelhante ao modelo de S. typhimurium. Além disso, a presença do gene oppA foi confirmada em todas as linhagens avaliadas. A OppA de várias espécies de Aeromonas foram reconhecidas por anticorpos obtidos contra a OppA de E. coli, confirmando a similaridade entre estas proteínas, e a expressão da OppA em Aeromonas. O seqüenciamento completo ou parcial do gene oppA de diferentes espécies de Aeromonas permitiu confirmar a elevada conservação do mesmo, e corroborar dados filogenéticos prévios. / Submitted by Marcelo Teixeira (mvteixeira@ucs.br) on 2014-05-22T17:08:12Z No. of bitstreams: 1 Dissertacao Fernanda Cattani.pdf: 1029794 bytes, checksum: cca1fad2170203cf94cc3056e3a6381b (MD5) / Made available in DSpace on 2014-05-22T17:08:12Z (GMT). No. of bitstreams: 1 Dissertacao Fernanda Cattani.pdf: 1029794 bytes, checksum: cca1fad2170203cf94cc3056e3a6381b (MD5) / The oligopeptide transport system (Opp system) is involved in different aspects of bacterial physiology, including nutrition, intercellular communication, and factors associated with virulence. These ABC transporters are formed by an oligopeptide binding protein, a permease, and ATP-binding domain. Aeromonas are ubiquous aquatic Gram-negative bacteria associated with several human diseases, particularly gastrointestinal disorders. Now a day, A. hydrophila, A. sobria and A. caviae are considered as emerging pathogens by the WHO. In this context, the objective of the present study was to characterize the oligopeptide transport system of Aeromonas using several bioinformatic and molecular tools. The results showed that as in other Gram-negative bacteria, the opp genes of Aeromonas are organized in a single policistronic operon formed by five genes (oppA, oppB, oppC, oppD and oppF). The oppA gene and its corresponding periplasmic oligopeptide-binding protein (OppA) are highly conserved, even between different bacterial families. A. hydrophila OppA model exhibits a typical Venus flytrap structure, similar to the S. typhimurium model. Furthermore, the presence of the oppA gene was confirmed in all the Aeromonas strains evaluated. The OppA of several Aeromonas species were recognized by antibodies obtained against E. coli OppA, confirming the similarity between these proteins, and the expression of the oligopeptide binding protein in Aeromonas. The complete or partial sequencing of the gene oppA of different species of Aeromonas allowed confirming the high conservation of this gene, and corroborate previous phylogenetic data.

O sistema de transporte de oligopeptídios (sistema Opp) está envolvido em diferentes aspectos da fisiologia bacteriana, incluindo nutrição, comunicação intercelular e fatores associados com a virulência. Estes transportadores ABC são formados por uma proteína de ligação a oligopeptídios, uma permease e um domínio de ligação ao ATP. As Aeromonas são bactérias Gram-negativas aquáticas ubíquas associadas com várias doenças em humanos, especialmente, gastrenterites. Atualmente, A. hydrophila, A. sobria e A. caviae são consideradas como patogenos emergentes pela OMS. Neste contexto, o objetivo do presente trabalho foi caracterizar o sistema de transporte de oligopeptídios em Aeromonas utilizando para tanto diversas ferramentas bioinformáticas e moleculares. Os resultados mostraram que, assim como em outras bactérias Gram-negativas, os genes opp de Aeromonas encontram-se organizados em um único operon policistrônico formado por cinco genes (oppA, oppB, oppC, oppD e oppF). O gene oppA e a proteína periplásmica de ligação a oligopeptídios correspondente (OppA) são altamente conservados, mesmo entre bactérias de famílias distintas. O modelo da proteína OppA de A. hydrophila mostrou a estrutura típica Venus flytrap , semelhante ao modelo de S. typhimurium. Além disso, a presença do gene oppA foi confirmada em todas as linhagens avaliadas. A OppA de várias espécies de Aeromonas foram reconhecidas por anticorpos obtidos contra a OppA de E. coli, confirmando a similaridade entre estas proteínas, e a expressão da OppA em Aeromonas. O seqüenciamento completo ou parcial do gene oppA de diferentes espécies de Aeromonas permitiu confirmar a elevada conservação do mesmo, e corroborar dados filogenéticos prévios. / The oligopeptide transport system (Opp system) is involved in different aspects of bacterial physiology, including nutrition, intercellular communication, and factors associated with virulence. These ABC transporters are formed by an oligopeptide binding protein, a permease, and ATP-binding domain. Aeromonas are ubiquous aquatic Gram-negative bacteria associated with several human diseases, particularly gastrointestinal disorders. Now a day, A. hydrophila, A. sobria and A. caviae are considered as emerging pathogens by the WHO. In this context, the objective of the present study was to characterize the oligopeptide transport system of Aeromonas using several bioinformatic and molecular tools. The results showed that as in other Gram-negative bacteria, the opp genes of Aeromonas are organized in a single policistronic operon formed by five genes (oppA, oppB, oppC, oppD and oppF). The oppA gene and its corresponding periplasmic oligopeptide-binding protein (OppA) are highly conserved, even between different bacterial families. A. hydrophila OppA model exhibits a typical Venus flytrap structure, similar to the S. typhimurium model. Furthermore, the presence of the oppA gene was confirmed in all the Aeromonas strains evaluated. The OppA of several Aeromonas species were recognized by antibodies obtained against E. coli OppA, confirming the similarity between these proteins, and the expression of the oligopeptide binding protein in Aeromonas. The complete or partial sequencing of the gene oppA of different species of Aeromonas allowed confirming the high conservation of this gene, and corroborate previous phylogenetic data.

La sécrétion de vésicules membranaires (VMs) constitue un processus physiologique important qui a particulièrement été étudié chez les Bactéries et les Eucaryotes. La récente découverte de la production de VMs chez les Archées souligne cependant que ce phénomène est universel et suggère que le dernier ancêtre commun aux trois domaines, LUCA (Last Universal Common Ancestor), produisait certainement des VMs. Les VMs des Archées n’ayant pour le moment été étudiées que chez certaines Crénarchées (ex : G/ Sulfolobus), nous avons entrepris de caractériser les VMs produites par un groupe d’Euryarchées hyperthermophiles anaérobies, les Thermococcales. Dans la première partie de cette étude, nous avons examiné le mécanisme de production ainsi que la composition en lipides et en protéines des VMs de trois espèces de Thermococcales: Thermococcus kodakaraensis, Thermococcus gammatolerans et Thermocococus sp. 5-4. Nous avons observé que les VMs sont sécrétées par un processus de bourgeonnement à partir de l’enveloppe cellulaire similaire à la formation des ectosomes par les cellules eucaryotes. De plus, les VMs sont fréquemment libérées en groupes, formant de grosses protubérances ou des filaments ressemblant aux nanopodes récemment décrits chez les Bactéries. Des différences de structure et de composition protéique sont observées entre les VMs des trois souches étudiées. Cependant, les VMs et les membranes cellulaires d’une même souche ont des compositions protéique et lipidique très proches, confirmant que les VMs sont produites à partir des membranes des cellules. Les VMs et les membranes cellulaires des trois souches comportent notamment un récepteur de peptides de la famille OppA (Oligopeptide-binding protein A) et des homologues de cette protéine ont été identifiés dans les VMs de certaines souches de Sulfolobus.Les VMs sécrétées par les Thermococcales sont associées à de l’ADN et cette association les protègent contre la thermodégradation. Nous montrons dans notre étude que les cellules de T. kodakaraensis transformées avec le plasmide navette plC70 relâchent des VMs comportant ce plasmide. De façon intéressante, ces VMs peuvent être utilisées pour transférer pLC70 à des cellules dénuées de plasmides, suggérant que les VMs pourraient être impliquées dans le transfert d’ADN entre cellules à haute température.Dans la seconde partie de cette étude, nous nous sommes particulièrement intéressés à la souche Thermococcus nautilus, une Thermococcale produisant des VMs associées de manière sélective à deux plasmides contenus dans la cellule. L’un d’eux correspond notamment à un génome viral défectueux de la lignée d’adenovirus PRD1. Ceci indique que les VMs peuvent être un moyen de transport pour des génomes viraux et suggère que la production de VMs par des cellules ancestrales pourraient avoir joué un rôle dans l’apparition des virus.En plus d’être impliquées dans le transport de plasmides/virus, les VMs produites par T. nautilus exercent un effet toxique sur certaines souches de Thermococcales, probablement dû au convoyage de toxines. Même si ces « thermococcines » nécessitent d’être caractérisées, il s’agit de la première mise en évidence d’une activité toxique liée aux VMs chez les Thermococcales. / Secretion of membrane vesicles (MVs) is an important physiological process that has been extensively studied in Bacteria and Eukarya. The recent discovery that Archaea produce MVs shows that this process is universal and suggests that the Last Universal Common Ancestor, LUCA, certainly produced MVs. As these archaeal MVs have been only studied in some Crenarchaeota (ex: G/ Sulfolobus), we started characterizing MVs produced by Thermococcales, a group of hyperthermophilic anaerobic Euryarchaeota.In the first part of this study we examined the mechanism of production as well as the protein and lipid composition of MVs produced by three strains of Thermococcales: Thermococcus kodakaraensis, Thermococcus gammatolerans and Thermocococus sp. 5-4. We observed that MVs are released by a budding process from the cell envelope that is similar to ectosome formation in eukaryotic cells. Moreover, clusters of MVs often form filamentous structures and protuberances on cell surfaces, resembling recently described bacterial nanopods. Differences in structure are observable between MVs of the three species, as well as in their protein composition. However, MVs and cell membranes from the same species have a quite similar protein and lipid composition, confirming that MVs are produced from cell membranes. A major protein present in cell membranes and MVs from the three strains is the oligopeptide-binding proteins (OppA), which has homologues in MVs from Sulfolobus species. Thermococcales MVs harbor DNA and protect this DNA against thermodegradation. Here, we show that T. kodakaraensis cells transformed with the shuttle plasmid pLC70 release MVs harboring this plasmid. Interestingly, these MVs can be used to transfer pLC70 into plasmid-free cells, suggesting that MVs could be involved in DNA transfer between cells at high temperature. In the second part of this study, we were specially interested in the strain Thermococcus nautilus, a Thermococcale that produces MVs selectively enriched in two plasmids from the cell. Notably, one of them corresponds to the genome of a defective virus from PRD1-adenovirus lineage. This indicates that MVs can be used as vehicles for the transport of viral genomes and suggests that production of MVs by ancestral cells could have played a role in the origin of viruses.In addition to be involved in transport of plasmids/viruses, MVs from T. nautilus display a toxic effect on some strains of Thermococcales, maybe due to the delivery of toxins. Even if these “thermococcins” remain to be characterized, this is the first time that a toxic activity associated with MVs has been shown in Thermococcales.

O sistema de captação de oligopeptideos (Opp), responsável pela captação de peptídeos com 3 ou mais resíduos de aminoácidos, representa um mecanismo importante de obtenção de nutrientes em bactérias. O operon Opp é constituído por 5 genes, sendo OppD e F responsáveis pela codificação dos componentes geradores, OppB e C codificantes para as proteínas que delimitam o poro da membrana e OppA que codifica o componente ligante. Neste trabalho identificamos uma alta identidade entre as proteínas OppA expressas por diferentes cepas de E. coli, 4 espécies do gênero Shigella ( 99 %) e diferentes sorovares de Salmonella enterica (85%) mas registramos a ocorrência de vários sítios polimórficos inter-específicos. A presença do gene OppA foi confirmada em 58 cepas diarreiogênicas de E. coli, Shigella e Salmonella. A partir da proteína OppA recombinante foi obtido soro policlonal específico que revelou a presença da proteína em todas as linhagens estudadas. Desta forma, concluímos que a proteína OppA está presente e conversada em espécies e linhagens dos três gêneros de Enterobacteriaceae estudados. / The oligopeptide uptake system (Opp), involved with the uptake of peptides formed by 3 or more amino acid residues, represent important nutrient uptake mechanism. The Opp operon is usually represented by 5 structural genes, including OppD and OppF encoding proteins involved generation of energy , OppB and OppC, encoding membrane proteins delimiting a pore and OppA encoding the protein responsible both for specificity and affinity of the transport system toward different peptide substrates. In this study, we demonstrated that the OppA proteins expressed by different E. coli strains,4 Shigella species (99%) and different serovars of Salmonella enterica (85%) were quite conserved but the occurrence of inter-species polymorphism was demonstrated. The OppA gene was detected in 58 diarrheogenic E. coli, Shigella and Salmonella strains. Using a recombinant OppA protein produced in E. coli, specific polyclonal sera were generated and successfully applied in the immunological detection of the proteins expressed by the tested strains. Thus, we conclude that the OppA protein is present and conserved among species and strains of the three test Enterobacteriaceae genera.

O sistema de captação de oligopeptideos (Opp), responsável pela captação de peptídeos com 3 ou mais resíduos de aminoácidos, representa um mecanismo importante de obtenção de nutrientes em bactérias. O operon Opp é constituído por 5 genes, sendo OppD e F responsáveis pela codificação dos componentes geradores, OppB e C codificantes para as proteínas que delimitam o poro da membrana e OppA que codifica o componente ligante. Neste trabalho identificamos uma alta identidade entre as proteínas OppA expressas por diferentes cepas de E. coli, 4 espécies do gênero Shigella ( 99 %) e diferentes sorovares de Salmonella enterica (85%) mas registramos a ocorrência de vários sítios polimórficos inter-específicos. A presença do gene OppA foi confirmada em 58 cepas diarreiogênicas de E. coli, Shigella e Salmonella. A partir da proteína OppA recombinante foi obtido soro policlonal específico que revelou a presença da proteína em todas as linhagens estudadas. Desta forma, concluímos que a proteína OppA está presente e conversada em espécies e linhagens dos três gêneros de Enterobacteriaceae estudados. / The oligopeptide uptake system (Opp), involved with the uptake of peptides formed by 3 or more amino acid residues, represent important nutrient uptake mechanism. The Opp operon is usually represented by 5 structural genes, including OppD and OppF encoding proteins involved generation of energy , OppB and OppC, encoding membrane proteins delimiting a pore and OppA encoding the protein responsible both for specificity and affinity of the transport system toward different peptide substrates. In this study, we demonstrated that the OppA proteins expressed by different E. coli strains,4 Shigella species (99%) and different serovars of Salmonella enterica (85%) were quite conserved but the occurrence of inter-species polymorphism was demonstrated. The OppA gene was detected in 58 diarrheogenic E. coli, Shigella and Salmonella strains. Using a recombinant OppA protein produced in E. coli, specific polyclonal sera were generated and successfully applied in the immunological detection of the proteins expressed by the tested strains. Thus, we conclude that the OppA protein is present and conserved among species and strains of the three test Enterobacteriaceae genera.

La sécrétion de vésicules membranaires (VMs) constitue un processus physiologique important qui a particulièrement été étudié chez les Bactéries et les Eucaryotes. La récente découverte de la production de VMs chez les Archées souligne cependant que ce phénomène est universel et suggère que le dernier ancêtre commun aux trois domaines, LUCA (Last Universal Common Ancestor), produisait certainement des VMs. Les VMs des Archées n'ayant pour le moment été étudiées que chez certaines Crénarchées (ex : G/ Sulfolobus), nous avons entrepris de caractériser les VMs produites par un groupe d'Euryarchées hyperthermophiles anaérobies, les Thermococcales. Dans la première partie de cette étude, nous avons examiné le mécanisme de production ainsi que la composition en lipides et en protéines des VMs de trois espèces de Thermococcales: Thermococcus kodakaraensis, Thermococcus gammatolerans et Thermocococus sp. 5-4. Nous avons observé que les VMs sont sécrétées par un processus de bourgeonnement à partir de l'enveloppe cellulaire similaire à la formation des ectosomes par les cellules eucaryotes. De plus, les VMs sont fréquemment libérées en groupes, formant de grosses protubérances ou des filaments ressemblant aux nanopodes récemment décrits chez les Bactéries. Des différences de structure et de composition protéique sont observées entre les VMs des trois souches étudiées. Cependant, les VMs et les membranes cellulaires d'une même souche ont des compositions protéique et lipidique très proches, confirmant que les VMs sont produites à partir des membranes des cellules. Les VMs et les membranes cellulaires des trois souches comportent notamment un récepteur de peptides de la famille OppA (Oligopeptide-binding protein A) et des homologues de cette protéine ont été identifiés dans les VMs de certaines souches de Sulfolobus.Les VMs sécrétées par les Thermococcales sont associées à de l'ADN et cette association les protègent contre la thermodégradation. Nous montrons dans notre étude que les cellules de T. kodakaraensis transformées avec le plasmide navette plC70 relâchent des VMs comportant ce plasmide. De façon intéressante, ces VMs peuvent être utilisées pour transférer pLC70 à des cellules dénuées de plasmides, suggérant que les VMs pourraient être impliquées dans le transfert d'ADN entre cellules à haute température.Dans la seconde partie de cette étude, nous nous sommes particulièrement intéressés à la souche Thermococcus nautilus, une Thermococcale produisant des VMs associées de manière sélective à deux plasmides contenus dans la cellule. L'un d'eux correspond notamment à un génome viral défectueux de la lignée d'adenovirus PRD1. Ceci indique que les VMs peuvent être un moyen de transport pour des génomes viraux et suggère que la production de VMs par des cellules ancestrales pourraient avoir joué un rôle dans l'apparition des virus.En plus d'être impliquées dans le transport de plasmides/virus, les VMs produites par T. nautilus exercent un effet toxique sur certaines souches de Thermococcales, probablement dû au convoyage de toxines. Même si ces " thermococcines " nécessitent d'être caractérisées, il s'agit de la première mise en évidence d'une activité toxique liée aux VMs chez les Thermococcales.