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

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal.

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal

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)

Streptococcus sanguinis is the second most common causative agent of bacterial infective endocarditis (IE). Risk of S. sanguinis IE is dependent on pre-disposing damage to the heart valve endothelium, which results in deposition of clotting factors for formation of a sterile thrombus (referred to as vegetation). Despite medical advances, high mortality and morbidity rates persist. Molecular characterization of S. sanguinis virulence determinants may enable development of prevention methods. In a previous screen for S. sanguinis virulence determinants by signature-tagged mutagenesis (STM) an attenuated mutant was identified with a transposon insertion in the nrdD gene, encoding an anaerobic ribonucleotide reductase. Evaluation of this mutant, as well as an nrdD in-frame deletion mutant, JFP27, by a soft-agar growth assay confirmed the anaerobic growth sensitivity of these strains. These studies suggest that an oxygen gradient occurs at the site of infection which selects for expression of anaerobic-specific genes at the nexus of the vegetation. The random STM screen failed to identify any favorable streptococcal surface-exposed prophylactic candidates. It was also apparent that additional genetic tools were required to facilitate the in vivo analyses of mutant strains. As it was desirable to insert antibiotic resistance markers into the chromosome, we identified a chromosomal site for ectopic expression of foreign genes. In vitro and in vivo analyses verified that insertion into this site did not affect important cellular phenotypes. The genetic tools developed facilitated further in vivo screening of S. sanguinis cell wall-associated (Cwa) protein mutants. A directed application of STM was employed for a comprehensive analysis of this surface protein class in the rabbit model of IE. Putative sortases, upon which Cwa proteins are dependent for cell surface localization, were also evaluated. No single S. sanguinis Cwa protein was determined essential for IE by STM screening; however competitiveness for colonization of the infection site was reduced for the mutant lacking expression of sortase A. The studies described here present a progressive picture of S. sanguinis IE, beginning with surface protein-dependent colonization of the vegetation in early IE, that later shifts to a bacterial persistence in situ dependent on condition-specific housekeeping genes, including nrdD.

Two Systems were designed. The first permits tunable on-demand softening of a hydrogel network. The second permits reversible on demand ligand exchange within a hydrogel network. Both means were shown to be cytocompatible and their uses demonstrated in cell culture of mesenchymal stem cells and 3T3 fibroblast cells.

Sortase A is a housekeeping enzyme of Gram-positive bacteria that catalyses the anchoring of surface proteins to the bacterial peptidoglycan. The enzyme works to establish an interaction between bacteria and host cells and is essential for pathogenesis. This makes Sortase A a potential suitable target for inhibition, in order to treat bacterial infections. In this degree project Sortase A from Staphylococcus aureus was explored and potential inhibitors were investigated by performing enzyme activity and bacterial binding assays. A robust FRET assay was developed and optimized for a recombinant version of the enzyme and serves as a good starting point for studying inhibition.

Staphylococcus aureus sortase A (SrtA) transpeptidase is a prominent membrane bound virulence factor in gram-positive bacteria, which organizes the peptidoglycan cell wall of the organism. Here, we report the first direct observation of the self-association behavior of SrtA. Formation of a SrtA dimer is highly selective in vitro in E. coli and in vivo on the S. aureus cell membrane. Quantitative analysis of protein binding affinity indicated a moderate association between two SrtA molecules with an apparent K[subscript d] of about 55 [micrometres] in vitro. Furthermore, to address the importance of dimerization for enzyme function, site-directed mutagenesis on potential target residues was performed to generate monomer only SrtA mutant proteins to completely disrupt dimer formation both in vitro and in vivo. Finally, an in vivo activity assay was performed to evaluate the function of SrtA wild type protein as well as its monomer only mutants. Our data demonstrated that S. aureus cells expressing mutant SrtA in a monomer only form are more successful at invading human epithelial cells than those expressing wild type SrtA in dimer-monomer equilibrium. It suggested that the monomeric form of SrtA is more active than the dimeric enzyme. We also demonstrated the uniqueness of SrtA dimerization by identifying that at least one other sortase family protein, SrtB only exists in monomer form. SrtA dimerization may have significant implications for understanding its biological function at both the cellular and molecular levels, which will lead to the development of new anti-infective therapies against gram-positive pathogens. / text

Proteases are important enzymes in the biotechnology due to their specific cleavage of substrates. HRV 3C, sortase A and TEV are some examples of cysteine proteases which become more of use lately in applications as removal of affinity tags (3C/TEV) and labelling of proteins (sortase). Here an investigation was made on the proteases by displaying them on two different prokaryotic hosts; E. coli and S. carnosus and to use these to cleave away affinity proteins (Affibody molecule) from other cells with an incorporated cleavage site. Constructs were cloned and incorporated into expressing strains which were then cultivated and induced. Analysis of surface expression was done by flow cytometer. Cleavage was made by cultivating combinations with cleavable bacteria and bacteria displaying proteases. A functional protease would lead to the presence of Affibody molecules in the supernatant. Flow cytomtery analysis was first made to inevstigate signal difference in Affibody binding by the addition of flurophores. Secondly SDS-PAGE was made on the centrifuged supernatant to investigate the presence of a product. Finally analysis of the bacteria was made by examining the reaction with soluble substrate and comparing activity with soluble enzyme. All of the enzymes were able to be displayed on the surface of bacteria with a clear separation from control. The cleavage analysis showed however varying results yet no clear evidence of product. Best flow cytometer results were seen for 3C but SDS-PAGE/MS did not show any cleaved product. For Sortase SDS-PAGE showed positive result but analysis with MS showed no product. TEV was concluded not to be funcional at all hence the failing to cleave soluble substrate  when condition seemed near optimal and faulty flow cytometer data. Even though the lack of success there is still many further studies that can be done on the proteases in order to prove its absence/presence  of activity.

Indiana University-Purdue University Indianapolis (IUPUI) / Dynamic changes to the extracellular matrix (ECM) impact many cell fate pro- cesses. The ECM can experience changes in sti ness as well as changes in composi- tion in response to injury, development, and diseases. To better understand the role that these dynamic processes have on the cells residing within the environment, re- searchers have turned towards 4-dimensional (4D) hydrogel designs. These 4D hydro- gels re-capitulate not only 3-dimensional (3D) matrix architectures, but also temporal changes in the physicochemical properties. The goal of this thesis was to design a unify chemistry (i.e., Sortase A (SrtA)-mediated transpeptidation) for dynamic tun- ing hydrogel sti ness and the presence of bioactive ligands. The rst objective was to establish a tunable and cytocompatible enzymatic scheme for softening cell-laden hydrogels. Brie y, the e ects of SrtA-mediated matrix cleavage were investigated us- ing poly(ethylene glycol) (PEG)-peptide hydrogels crosslinked by SrtA-sensitive and insensitive peptides. Initially, the e ects of various parameters with respect to cat- alytic reactions of SrtA were characterized rheologically, including enzyme and sub- strate concentrations, macromer content, peptide composition, and treatment time. Gel moduli pre- and post-enzyme treatment were measured to verify SrtA-mediated hydrogel softening. The cytocompatibility of SrtA-mediated gel softening system was investigated using human mesenchymal stem cell (hMSC). Upon treatment with SrtA and an oligoglycine substrate, encapsulated hMSCs exhibited extensive spreading in comparison to those within statically sti matrices. The second objective was to es- tablish a reversible ligand exchange system utilizing SrtA-mediated transpeptidation. SrtA-sensitive pendant ligands were immobilized within PEG hydrogels, which were treated with SrtA and an oligoglycine substrate to a ord tunable removal of the pen- dant ligand. Through measurement of the liberated pendant peptide concentration, it was found that higher concentrations of SrtA or extending treatment times led to higher ligand removal e ciency. Finally, the e ect of peptide ligand removal on cell behaviors were evaluated using NIH 3T3 broblasts. Fibroblasts were culture both on and within hydrogels containing SrtA-cleavable cell adhesion peptide. After treatment, both conditions led to a decrease in broblast spreading in comparison to non-treated gels. Overall, the utility of SrtA as versatile agent for controlling the mechanical properties and the presence of biologically active components within a hydrogel system was demonstrated. These systems could be further explored with natural-based materials to better mimic the physiological environment experienced by cells.