Current treatment methods for non-union bone defects present problems. The objective of this study was to genetically engineer primary and immortalized cell types to express osteogenic molecules BMP2, RUNX2, OSX, or VEGF in a doxycycline dose-dependent manner for tissue regeneration. Coding cDNA sequences for all four factors were sub-cloned into the pRTS-1 expression plasmid and transfected into HUCPVCs, RBMCs, ROS cells. Electroporation was the most effective method of transfection for all cells but stably transfected cells could only be established for RBMCs and ROS cells. Cells achieved maximum expression within 72hours of induction and returned to basal levels after 18 days. Enhanced osteogenic bioactivity was only observed upon activation of BMP-2. The tight regulation of the pRTS-1 system allowed for a controlled gene expression. Future transplantation experiments using these engineered RBMC and ROS cells in vivo will evaluate the usefulness of the dox-inducible gene expression system in bone defects.

Current treatment methods for non-union bone defects present problems. The objective of this study was to genetically engineer primary and immortalized cell types to express osteogenic molecules BMP2, RUNX2, OSX, or VEGF in a doxycycline dose-dependent manner for tissue regeneration. Coding cDNA sequences for all four factors were sub-cloned into the pRTS-1 expression plasmid and transfected into HUCPVCs, RBMCs, ROS cells. Electroporation was the most effective method of transfection for all cells but stably transfected cells could only be established for RBMCs and ROS cells. Cells achieved maximum expression within 72hours of induction and returned to basal levels after 18 days. Enhanced osteogenic bioactivity was only observed upon activation of BMP-2. The tight regulation of the pRTS-1 system allowed for a controlled gene expression. Future transplantation experiments using these engineered RBMC and ROS cells in vivo will evaluate the usefulness of the dox-inducible gene expression system in bone defects.

Photorhabdus é um gênero de enterobactérias simbiontes de Heterorhabditis, um gênero de nematoides entomopatogênicos. Dentre as enzimas secretadas por P. luminescens TTO1, destaca-se PrtA, uma metaloprotease que pertence a subfamília das serralisinas. PrtS, uma protease capaz de induzir uma forte resposta de melanização no inseto, foi identificada em P. temperata Az29 mas não em P. luminescens TTO1. Neste trabalho foram detectadas e caracterizadas bioquimicamente algumas proteases secretadas pelo isolado MN7 de P. luminescens luminescens. Foram detectadas duas atividades mais proeminentes: uma de 50 kDa, e outra de 38 kDa. Com o sequenciamento do genoma desta bactéria, pudemos confirmar a presença no genoma de genes codificando proteínas de alta identidade com as descritas PrtA e PrtS, de massas similares às detectadas nas nossas zimografias. As proteases são inibidas por inibidores específicos de metaloproteases. P. luminescens MN7 apresenta secreção, portanto, de uma protease já descrita algumas vezes, e de outra presente apenas em P. temperata Az29. / Photorhabdus is a genus of Enterobacteriaceae, symbionts of Heterorhabditis, a genus of entomopathogenic nematodes. Among the enzymes secreted by P. luminescens TTO1 stands out PrtA, a metalloprotease that belongs to the subfamily of serralysins. PrtS, a protease capable of inducing a strong melanotic response from the insect, was identified in P. temperata Az29 but not in P. luminescens TTO1. In this work were detected and characterized biochemically few isolated proteases secreted by P. luminescens luminescens MN7. Two most prominent activities were detected: one with 50 kDa and one with 38 kDa. With the sequencing of the genome of MN7, we could confirm the presence in the genome of genes encoding proteins with high identity to PrtA and PrtS already described, with similar masses to those detected in our zimographies. These proteases are inhibited by specific inhibitors of metalloproteases. P. luminescens MN7 secretes a protease already described a few times, and other present only in P. temperata Az29.

Nous nous intéressons à la production de peptides bioactifs dans des laits fermentés par la bactérie lactique Streptococcus thermophilus. Pour ce faire, il est nécessaire que cette bactérie en internalise le moins possible lors de sa croissance. Il était donc important de caractériser le système protéolytique S. thermophilus. Tout d’abord, les relations phylogéniques liant 30 souches de S. thermophilus ont été recherchées par MLST. Ensuite, un système de transport de type ABC qui semble fonctionnel a été identifié chez la souche LMD-9 et appelé OTS. Une étude de la variabilité des systèmes de transport Ami et OTS des 30 souches de S. thermophilus a été réalisée. Enfin, l’hydrolyse des caséines par la protéase PrtS de S. thermophilus a été étudiée. Cette protéase habituellement ancrée à la paroi de la bactérie est retrouvée chez la souche 4F44 également sous forme libre. La séquence protéique de PrtS4F44, différente de celle de PrtS de la souche LMD 9 (PrtSLMD-9), n’est pas la cause de la libération partielle de PrtS4F44. La sortase A, acteur de l’ancrage de PrtS à la paroi de la bactérie, présente chez la souche 4F44 (srtA4F44) un allèle différent de celui de la souche LMD-9 (srtALMD-9). En effet, PrtSLMD-9 se trouve libérée lorsque srtALMD-9 est remplacée par srtA4F44 dans la souche LMD-9 montrant ainsi que SrtA4F44 est déficiente, entrainant par conséquent un défaut d’ancrage de PrtS4F44 et sa libération partielle dans le milieu extracellulaire. L’hydrolyse des caséinates bovines totales par la forme libre de PrtS4F44 a permis d’obtenir des peptides bioactifs qui pourront être utilisés pour la fonctionnalisation de produits laitiers fermentés / We are interested in the production of bioactive peptides in fermented milk by the lactic acid bacterium Streptococcus thermophilus. For this, it requires that the bacterium internalize them as few as possible during its growth. Therefore, it was important to characterize the proteolytic system of S. thermophilus. First, phylogenetic relationships linking 30 S. thermophilus strains have been searched by MLST. Secondly, an ABC-type transport system which seems to be functional was identified in the LMD-9 strain and named OTS. A study of the variability of Ami and OTS transport systems of the 30 strains of S. thermophilus was performed. Finally, the hydrolysis of caseins by proteinase PrtS of S. thermophilus was studied. This proteinase usually anchored to the wall of the bacterium was also found in a free form in strain 4F44. The protein sequence of PrtS4F44, different from the one of PrtS in the LMD-9 strain (PrtSLMD-9), is not the cause of the partial release of PrtS4F44. Sortase A, the actor of the anchoring of PrtS to the wall of the bacteria, presents different alleles between the strain 4F44 (srtA4F44) and the LMD-9 strain (srtALMD-9). Indeed, PrtSLMD-9 is released when srtALMD-9 is replaced by srtA4F44 in the strain LMD-9 showing that SrtA4F44 is deficient, causing consequently a default of PrtS4F44 anchoring and its partial release into the extracellular medium. Additionally, hydrolysis of bovine caseinates was performed using the free form PrtS4F44 and allowed the production of bioactive peptides that can be used for the functionalization of fermented dairy products

Photorhabdus é um gênero de enterobactérias simbiontes de Heterorhabditis, um gênero de nematoides entomopatogênicos. Dentre as enzimas secretadas por P. luminescens TTO1, destaca-se PrtA, uma metaloprotease que pertence a subfamília das serralisinas. PrtS, uma protease capaz de induzir uma forte resposta de melanização no inseto, foi identificada em P. temperata Az29 mas não em P. luminescens TTO1. Neste trabalho foram detectadas e caracterizadas bioquimicamente algumas proteases secretadas pelo isolado MN7 de P. luminescens luminescens. Foram detectadas duas atividades mais proeminentes: uma de 50 kDa, e outra de 38 kDa. Com o sequenciamento do genoma desta bactéria, pudemos confirmar a presença no genoma de genes codificando proteínas de alta identidade com as descritas PrtA e PrtS, de massas similares às detectadas nas nossas zimografias. As proteases são inibidas por inibidores específicos de metaloproteases. P. luminescens MN7 apresenta secreção, portanto, de uma protease já descrita algumas vezes, e de outra presente apenas em P. temperata Az29. / Photorhabdus is a genus of Enterobacteriaceae, symbionts of Heterorhabditis, a genus of entomopathogenic nematodes. Among the enzymes secreted by P. luminescens TTO1 stands out PrtA, a metalloprotease that belongs to the subfamily of serralysins. PrtS, a protease capable of inducing a strong melanotic response from the insect, was identified in P. temperata Az29 but not in P. luminescens TTO1. In this work were detected and characterized biochemically few isolated proteases secreted by P. luminescens luminescens MN7. Two most prominent activities were detected: one with 50 kDa and one with 38 kDa. With the sequencing of the genome of MN7, we could confirm the presence in the genome of genes encoding proteins with high identity to PrtA and PrtS already described, with similar masses to those detected in our zimographies. These proteases are inhibited by specific inhibitors of metalloproteases. P. luminescens MN7 secretes a protease already described a few times, and other present only in P. temperata Az29.

Parmi 30 souches, seule 4F44 excrète une activité dans son milieu de culture qui ne résulte pas de la présence de protéases intracellulaires due à une lyse cellulaire.D’après le séquençage N-ter, l’enzyme soluble chez 4F44 est PrtS retrouvée sous deux formes (non mature et mature) à la fois ancrée (60%) et soluble (40%).Sa séquence protéique déduite de prtS est proche de celles de LMD-9 (97% identité), CNRZ385 (98%), JIM8232 (96%) et S. suis (97%) chez qui elle est toujours ancrée. Le domaine d’ancrage contenant LPNTG est conservé chez 4F44, LMD-9, CNRZ385 et S. suis ; ainsi elle serait ancrée au peptidoglycane par la sortase A (SrtA). Chez 4F44, l’absence d’une duplication peptidique imparfaite dans le prodomaine de PrtS pourrait expliquer sa libération partielle, en effet LMD-9 présentant cette duplication possède sa protéase PrtS uniquement sous forme ancrée.La comparaison de la séquence de la sortase déduite du gène de 4F44, ND03, LMD-9, PB18O, PB302 et CNRZ307 a montré que les sites catalytique et actif sont conservés. Seuls 6 résidus d’acides aminés sont différents, la substitution chez 4F44 de I222 par V222 entraînerait sa libération partielle. Les peptides trypsiques C-ter QVTQLPNTGENDTK et QVTQLPNTGENDTKYYLVPGVIIGLGTLLVSIRR ont été identifiés en MS/MS, donc la liaison TG (cible de l’activité peptidasique de SrtA) n’est pas hydrolysée. La caséine β est la caséine préférentiellement hydrolysée. L’enzyme présente une large spécificité de coupure (A, L, M, F, Y, W, V, S, T, N, Q, R, H et K en position P1). Globalement elle libère 33 peptides bioactifs : ECA-inhibiteurs, mitogènes, opioïdes, immunomodulants, antibactériens, antioxydants, antimutagènes / Among 30 strains, only 4F44 strain releases a activity in the medium which does not results from the presence of intracellular protease due to cell lysis. This soluble protease is PrtS in two forms (non mature and mature), 60% as anchored to cell wall and 40% as released in the medium. The protein sequence deduced from the gene is slightly different to those of LMD-9 (97% identity), CNRZ385 (98%), JIM8232 (96%), S. suis (97%). The protein sequence of the anchor domain including LPNTG is conserved as for LMD-9, CNRZ385 and S. suis ; so this PrtS might be anchored to peptidoglycan by sortase A (SrtA).In 4F44, the absence of an imperfect duplication of a peptide sequence at prodomain of PrtS could explain the partial liberation, furthermore, the LMD-9 strain which presents this duplication possess the protease PrtS only the anchored form.Comparison of protein sequence deduced from the srtA gene in 4F44, ND03, LMD-9, PB18O, PB302 and CNRZ307 strains showed that the residues of the catalytic and active sites are conserved. Six amino acids are different for 4F44, I222 replaced by a V222 possibly leads to the partial liberation of PrtS. The trypsic C-ter peptides QVTQLPNTGENDTK and QVTQLPNTGENDTKYYLVPGVIIGLGTLLVSIRR have been identified by MS/MS, indicating that the peptide bond of T and G (target of endopeptidasic activity of Srt A) is not hydrolyzed.β-casein is preferentially degraded in comparison to other caseins. Soluble PrtS has a broad specificity against A, L, M, F, Y, W, V, S, T, N, Q, R, H and K at position P1. Globally, it releases 33 bioactive peptides (antihypertensives, mitogenics, oipoide, immunomodulantors, antimicrobials, antioxydants, antimutagens)