<p>Abstract Redacted.</p> / Master of Science (MSc)

Candida albicans est l'espèce la plus communément identifiée dans les pathologies fongiques buccales. Les traitements des infections à C. albicans se font par le biais d'antifongiques. L'efficacité des antifongiques est cependant limitée aux formes prolifératives et non aux formes stationnaires de C. albicans. De plus, plusieurs cas de résistance de C. albicans aux antifongiques ont été rapportés. Par conséquent, trouver une nouvelle molécule antifongique a toute son importance pour le traitement des cas de candidoses. Le but de cette étude est d'évaluer l'activité fongicide de la nisine Z. Pour ce faire, nous avons analysé l'effet de la nisine sur l'inhibition de C. albicans en utilisant la méthode de l' "Alamar blue". L'efficacité de la nisine Z, à bloquer la transformation de C. albicans de la forme blastospore à la forme hyphe (plus virulente), a été évaluée par observation microscopique et par dénombrement des différentes formes. Nos résultats montrent que la nisine Z inhibe la croissance de C. albicans avec des pourcentages d'inhibition de 70, 63 et 53 % pour des concentrations respectives de 1000, 500 et 100 mu/ml. Cependant, seule la concentration de 1000 mu/ml permet de réduire de 45 % la transformation de C. albicans par rapport au contrôle. Ces résultats sont confirmés par l'observation microscopique. En conclusion, la nisine Z réduit la prolifération et la transformation de C. albicans, suggérant sa potentielle utilisation pour le traitement de.s candidoses.

L'objectif du présent projet était de trouver des bactéries lactiques épiphytes des plantes fourragères qui produisent des bactériocines avec activité anti-clostridiale. Enterococcus sp. 828 a été retenu pour des essais d'ensilement en laboratoire avec du matériel végétal irradié et dans des silos à échelle pilote. Tous les ensilages étaient inoculés avec des spores de Clostridium tyrobutyricum ATCC 25755. Lactococcus lactis ATCC 14545, qui produit la nisine A, a servi de témoin positif. Seule la nisine A a été détectée dans les essais d'ensilement en laboratoire. Dans les silos à échelle pilote, une faible production de substances inhibitrices a peut-être été observée dans certains cas. C. tyrobutyricum ATCC 25755 s'est peu développé dans les silos témoin ou inoculés. Par conséquent, aucune diminution du nombre de C. tyrobutyricum par les deux bactéries antagonistes n'a pu être observée dans les deux types de silos. Enterococcus sp. 828 et L. lactis ATCC 14545 semblent peu compétitives en présence d'une population épiphyte.

Pour produire des ferments lactiques contenant des bifidobactéries par des cellules immobilisées, des fermentations ont été réalisées avec une culture pure de bifidobactéries ou en culture mixte avec une souche de lactocoques. Une méthode a été développée pour localiser et quantifier par immunofluorescence les deux souches dans des billes de gel. L'utilisation de cette méthode a permis d'observer une croissance bactérienne préférentielle à la périphérie des billes. La production continue de culture mixte a été étudiée avec un système composé d'un premier réacteur (R1) contenant les deux souches immobilisées séparément dans des billes de gel et d'un second réacteur (R2) opéré avec les cellules libres relarguées de R1. Une culture mixte concentrée de composition stable a été produite à 35ʻC dans l'effluent de R2. La redistribution des souches dans les billes fut observée en microscopie confocale. La résistance à différents stress des cellules libres produites dans l'effluent des deux réacteurs a augmenté avec le temps de fermentation.

La régulation de l’expression génique chez les bactéries résulte d’un processus complexe comprenant deux étapes majeures, la transcription des gènes en ARNm et leur traduction en protéines. Les études qui allient les données de transcription et de traduction sont rares et l’importance de chacun de ces deux mécanismes dans un processus global d’adaptation n’est pas encore clairement définie. Or, les faibles corrélations entre les niveaux d’ARNm et de protéines chez les bactéries et, plus particulièrement chez la bactérie modèle Lactococcus lactis, suggèrent l’importance des régulations traductionnelles.Aujourd’hui des exemples de mécanismes de régulation de la traduction à l’échelle moléculaire se multiplient, néanmoins il n’existe que très peu de méthodes systémiques permettant d’étudier ces régulations à l’échelle globale. Dans cette thèse, l’état de traduction de chacun des ARNm de la cellule a été estimé par la mesure du traductome. Ainsi, pour chaque ARNm, le pourcentage de molécules en traduction et sa densité en ribosomes ont été déterminés. Pour la première fois, une image complète de l’état de traduction de la bactérie a été obtenue montrant une grande variabilité traductionnelle au sein de la population des transcrits. De plus, il a été démontré que cet état traductionnel était très régulé. De fait, lors d’une carence nutritionnelle, la machinerie de traduction est globalement diminuée et il est observé une redistribution de l’efficacité de traduction vers des gènes nécessaires à la bactérie pour être adaptée au stress imposé. D’autre part, cette forte variabilité de l’état de traduction au sein des ARNm a pu être reliée à des différences au niveau du mécanisme propre de la traduction. En effet, les coefficients de contrôle des trois grandes étapes de la traduction, estimés par modélisation à partir des données de traductome, dépendent fortement de la nature des gènes. Ainsi un contrôle au niveau de l’étape d’initiation a été démontré comme attendu pour la majorité des gènes. Mais pour un grand nombre de gènes, un contrôle par l’élongation (et pour un nombre plus restreint par la terminaison) a été aussi mis en évidence chez L. lactis. Dans le contrôle global de l’expression génique, il a d’autre part été mis en évidence que les processus de traduction et de dégradation des ARNm étaient impliqués et associés à des régulations coordonnées ou non en fonction des conditions de croissance.En conclusion, ces travaux de thèse ont montré l’importance des régulations de la traduction. Plus largement, ils ont souligné la nécessité de caractériser les différents niveaux de régulations de l’expression génique afin de mieux appréhender la physiologie de la cellule / In bacteria, regulation of gene expression results from a complex program composed of two main steps: transcription of genes into mRNA and their translation into proteins. Few studies integrate both transcription and translation, so their relative importance in the global process of bacterial adaptation is not yet well defined. However, weak correlations between mRNA and protein levels were found in bacteria, in particular in the lactic acid bacteria model Lactococcus lactis, suggesting significant translation regulations in this bacterium.Nowadays, translation regulation mechanisms are mainly investigated at the molecular level since only few systemic methods exist to study these regulations at a genome-wide scale. During this PhD, translation state of all mRNA was estimated by translatome measurement. For each mRNA in the cell, percentage of its molecules in translation and its ribosome density were determined. For the first time in bacteria, a detailed picture of the translation state of all transcripts was obtained. Large variation of translation state was observed within the transcript population demonstrating a high diversity of translational regulations in a given physiological state. In addition, during nutrient starvation, the global translation machinery was decreased and associated with a redistribution of the translation efficiency towards genes required to stress adaptation.Changes in translation state were related to specific kinetics of the three elementary steps of translation. From translatome data, control coefficients of initiation, elongation and termination on the global translation process were modeled. The translation limiting step was strongly dependent on gene function. Although a control by initiation was observed for most of the genes of L. lactis, a large set of genes was elongation limited, and even few genes were limited by termination.In the global control of gene expression, both translation and mRNA decay were involved and led to coupled or uncoupled regulations according to growth conditions.Finally, this work has demonstrated the importance of translation regulations in bacteria. This result strengthens the necessity to include all the different layers of gene expression regulation in order to better understand cell physiology

The present study aimed to evaluate bacterial isolates and the microbiome of dairies. The specific aims were: to characterize Staphylococcus spp. isolated from mastitic milk, to evaluate the presence of Lactococcus in mastitic milk as a potential causative agent of mastitis, to evaluate the association between microbiome and milk quality parameters, and to characterize Staphylococcus spp. isolated from production lines of Minas Frescal cheese. The detection of genes encoding virulence factors (enterotoxins (sea, seb, sec, sed, see, seg, seh, sei, selj, selk, sell, selm, seln, selo, selp, seIq, ser, ses, set, selu, selv, and selx), hemolysins (hla, hlb, hld, hlg, and hlgv), exfoliative toxins (eta, etb, and etd), Panton-Valentine leukocidin (pvl), and toxic shock syndrome toxin (tst)), genes encoding antibiotic resistance (resistance to tetracycline (tetK, tetL, and tetM), erythromycin (ermA, ermB, and ermC), methicillin (mecA and mecC), and tobramycin (ant(4\')-Ia)), molecular typing (spa, SCCmec, and agr types), and phenotyping regarding antibiotic resistance were performed in staphylococci isolates from mastitic milk, and from cheese processing plant samples. Staphylococcus aureus was identified in the majority of isolates from both origins. Several virulence factor genes were detected. The distribution of genes encoding staphylococcal enterotoxins (85.0% - 85.7% of isolates were positive for one or more enterotoxin gene) was highlighted and the gene related to H toxin was the most prevalent. Methicillin-resistant Staphylococcus aureus were identified in isolates from mastitic milk (4.1%) and cheese processing (6.0%); the genotyping and phenotyping of these isolates were described. t605 had the highest frequency in the S. aureus population studied. In mastitic milk, Lactococcus was suggested as the causative agent of an outbreak of mastitis in a dairy farm. Using next generation sequencing, the abundance of Lactococcus was observed in microbiome samples. Bacterial isolation and DNA sequencing confirmed the presence of Lactococcus lactis and Lactococcus garvieae. The microbiome of environmental samples and bulk tank milk from the dairy farm showed the Lactococcus genus among the most common bacterial taxa, suggesting other sources of this genus. Regarding milk quality parameters, the microbiome of bulk tank milk from several dairy farms was associated with somatic cell count and bacterial count. The core microbiome was described and many genera of importance were identified. Among the associations performed between microbiome and milk quality parameters, the identification of Streptococcus in samples classified with high somatic cell count and high bacterial count was highlighted. Several bacterial taxa with relative abundance significantly higher in samples classified as high and low cell count and bacterial count were shown. Real-time polymerase chain reaction was also performed associated with bacterial diversity, bacterial taxa, and bacterial count. These findings highlight the need to control and prevent bacterial contamination in the dairy industry, from herd to consumers. / O presente estudo apresentou como objetivo avaliar isolados bacterianos e microbioma de lácteos. Os objetivos específicos foram: caracterizar Staphylococcus spp. isolados de leite de vacas com mastite, avaliar a presença de Lactococcus em leite de vacas com mastite como um potencial agente causador de mastite, avaliar a associação entre microbioma de leite de tanque e parâmetros da qualidade de leite, e caracterizar Staphylococcus spp. isolados de linhas de processamento de queijo Minas frescal. A detecção de genes codificadores de fatores de virulência (enterotoxinas (sea, seb, sec, sed, see, seg, seh, sei, selj, selk, sell, selm, seln, selo, selp, seIq, ser, ses, set, selu, selv, e selx), hemolisinas (hla, hlb, hld, hlg, e hlgv), toxinas exfoliativas (eta, etb e etd), leucocidina de Panton-Valentine (pvl), toxina da síndrome do choque tóxico (tst)), genes codificadores de resistência a antibióticos (resistência a tetraciclina (tetK, tetL e tetM), eritromicina (ermA, ermB e ermC), meticilina (mecA e mecC) e tobramicina (ant(4\')-Ia)), tipagem molecular (spa, SCCmec e agr types), e fenotipagem quanto à resistência a antibióticos foram realizadas em estafilococos isolados de leite de vacas com mastite e de amostras de planta de processamento de queijo. Staphylococcus aureus foi identificado na maioria dos isolados de ambas as origens. Diversos genes de fatores de virulência foram detectados, com destaque para a distribuição de genes codificadores de enterotoxinas estafilocócicas (85,0%-85,7% dos isolados foram positivos para um ou mais genes codificadores de enterotoxinas), sendo o gene relacionado com a toxina H o mais frequente. Staphylococcus aureus meticilina resistente foram identificados em isolados de leite de vacas com mastite (4.1%) e em processamento de queijo (6.0%); o perfil genotípico e fenotípico destes isolados foram descritos. t605 foi o mais freqüente na população de S. aureus estudada. Em leite de vacas com mastite, Lactococcus foi sugerido como o agente causador de um surto de mastite numa fazenda leiteira. Usando sequenciamento de nova geração, a abundância de Lactococcus foi observada no microbioma das amostras. O isolamento e sequenciamento de DNA confirmaram a presença de Lactococcus lactis e Lactococcus garvieae. O microbioma de amostras ambientais e de leite de tanque da fazenda mostrou o gênero Lactococcus entre os mais comuns, sugerindo outras fontes deste gênero. Contemplando parâmetros da qualidade de leite, o microbioma de leite de tanque de várias fazendas leiteiras foi relacionado com contagem de células somáticas e contagem bacteriana. O core microbiome foi descrito e muitos gêneros bacterianos de importância foram identificados. Dentre as análises realizadas associando microbioma com parâmetros da qualidade de leite, foi destacada a identificação de Streptococcus em amostras classificadas com alta contagem de células somáticas e alta contagem bacteriana. Diversos táxons bacterianos com abundância relativa significativamente maior em amostras classificadas com alta e baixa contagem de células somáticas e contagem bacteriana foram mostrados. Reação em cadeia da polimerase em tempo real também foi realizada e associada com diversidade bacteriana, táxons bacterianos e contagem bacteriana. Estes levantamentos confirmam a necessidade de controlar e prevenir a contaminação bacteriana na indústria de lácteos, do rebanho leiteiro até os consumidores.

The present study aimed to evaluate bacterial isolates and the microbiome of dairies. The specific aims were: to characterize Staphylococcus spp. isolated from mastitic milk, to evaluate the presence of Lactococcus in mastitic milk as a potential causative agent of mastitis, to evaluate the association between microbiome and milk quality parameters, and to characterize Staphylococcus spp. isolated from production lines of Minas Frescal cheese. The detection of genes encoding virulence factors (enterotoxins (sea, seb, sec, sed, see, seg, seh, sei, selj, selk, sell, selm, seln, selo, selp, seIq, ser, ses, set, selu, selv, and selx), hemolysins (hla, hlb, hld, hlg, and hlgv), exfoliative toxins (eta, etb, and etd), Panton-Valentine leukocidin (pvl), and toxic shock syndrome toxin (tst)), genes encoding antibiotic resistance (resistance to tetracycline (tetK, tetL, and tetM), erythromycin (ermA, ermB, and ermC), methicillin (mecA and mecC), and tobramycin (ant(4\')-Ia)), molecular typing (spa, SCCmec, and agr types), and phenotyping regarding antibiotic resistance were performed in staphylococci isolates from mastitic milk, and from cheese processing plant samples. Staphylococcus aureus was identified in the majority of isolates from both origins. Several virulence factor genes were detected. The distribution of genes encoding staphylococcal enterotoxins (85.0% - 85.7% of isolates were positive for one or more enterotoxin gene) was highlighted and the gene related to H toxin was the most prevalent. Methicillin-resistant Staphylococcus aureus were identified in isolates from mastitic milk (4.1%) and cheese processing (6.0%); the genotyping and phenotyping of these isolates were described. t605 had the highest frequency in the S. aureus population studied. In mastitic milk, Lactococcus was suggested as the causative agent of an outbreak of mastitis in a dairy farm. Using next generation sequencing, the abundance of Lactococcus was observed in microbiome samples. Bacterial isolation and DNA sequencing confirmed the presence of Lactococcus lactis and Lactococcus garvieae. The microbiome of environmental samples and bulk tank milk from the dairy farm showed the Lactococcus genus among the most common bacterial taxa, suggesting other sources of this genus. Regarding milk quality parameters, the microbiome of bulk tank milk from several dairy farms was associated with somatic cell count and bacterial count. The core microbiome was described and many genera of importance were identified. Among the associations performed between microbiome and milk quality parameters, the identification of Streptococcus in samples classified with high somatic cell count and high bacterial count was highlighted. Several bacterial taxa with relative abundance significantly higher in samples classified as high and low cell count and bacterial count were shown. Real-time polymerase chain reaction was also performed associated with bacterial diversity, bacterial taxa, and bacterial count. These findings highlight the need to control and prevent bacterial contamination in the dairy industry, from herd to consumers. / O presente estudo apresentou como objetivo avaliar isolados bacterianos e microbioma de lácteos. Os objetivos específicos foram: caracterizar Staphylococcus spp. isolados de leite de vacas com mastite, avaliar a presença de Lactococcus em leite de vacas com mastite como um potencial agente causador de mastite, avaliar a associação entre microbioma de leite de tanque e parâmetros da qualidade de leite, e caracterizar Staphylococcus spp. isolados de linhas de processamento de queijo Minas frescal. A detecção de genes codificadores de fatores de virulência (enterotoxinas (sea, seb, sec, sed, see, seg, seh, sei, selj, selk, sell, selm, seln, selo, selp, seIq, ser, ses, set, selu, selv, e selx), hemolisinas (hla, hlb, hld, hlg, e hlgv), toxinas exfoliativas (eta, etb e etd), leucocidina de Panton-Valentine (pvl), toxina da síndrome do choque tóxico (tst)), genes codificadores de resistência a antibióticos (resistência a tetraciclina (tetK, tetL e tetM), eritromicina (ermA, ermB e ermC), meticilina (mecA e mecC) e tobramicina (ant(4\')-Ia)), tipagem molecular (spa, SCCmec e agr types), e fenotipagem quanto à resistência a antibióticos foram realizadas em estafilococos isolados de leite de vacas com mastite e de amostras de planta de processamento de queijo. Staphylococcus aureus foi identificado na maioria dos isolados de ambas as origens. Diversos genes de fatores de virulência foram detectados, com destaque para a distribuição de genes codificadores de enterotoxinas estafilocócicas (85,0%-85,7% dos isolados foram positivos para um ou mais genes codificadores de enterotoxinas), sendo o gene relacionado com a toxina H o mais frequente. Staphylococcus aureus meticilina resistente foram identificados em isolados de leite de vacas com mastite (4.1%) e em processamento de queijo (6.0%); o perfil genotípico e fenotípico destes isolados foram descritos. t605 foi o mais freqüente na população de S. aureus estudada. Em leite de vacas com mastite, Lactococcus foi sugerido como o agente causador de um surto de mastite numa fazenda leiteira. Usando sequenciamento de nova geração, a abundância de Lactococcus foi observada no microbioma das amostras. O isolamento e sequenciamento de DNA confirmaram a presença de Lactococcus lactis e Lactococcus garvieae. O microbioma de amostras ambientais e de leite de tanque da fazenda mostrou o gênero Lactococcus entre os mais comuns, sugerindo outras fontes deste gênero. Contemplando parâmetros da qualidade de leite, o microbioma de leite de tanque de várias fazendas leiteiras foi relacionado com contagem de células somáticas e contagem bacteriana. O core microbiome foi descrito e muitos gêneros bacterianos de importância foram identificados. Dentre as análises realizadas associando microbioma com parâmetros da qualidade de leite, foi destacada a identificação de Streptococcus em amostras classificadas com alta contagem de células somáticas e alta contagem bacteriana. Diversos táxons bacterianos com abundância relativa significativamente maior em amostras classificadas com alta e baixa contagem de células somáticas e contagem bacteriana foram mostrados. Reação em cadeia da polimerase em tempo real também foi realizada e associada com diversidade bacteriana, táxons bacterianos e contagem bacteriana. Estes levantamentos confirmam a necessidade de controlar e prevenir a contaminação bacteriana na indústria de lácteos, do rebanho leiteiro até os consumidores.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / The objective of this study was to produce and to test the performance of native starters cultures in the elaboration of fermented Italian sausage in relation to the security and quality of the sausages. In the first experiment strains of Staphylococcus xylosus isolated from colonial sausages, were characterized and after identified for the Kit Api Staph (Biomérieux). In the second experiment, the microorganism Lactococcus lactis ssp lactis was fermented in pork plasma culture medium and evaluated its efficiency in relation to MRS broth, through microbiological analyses and optic density (OD). In the third experiment the isolated and multiplied cultures were added in dry fermented sausages, elaborating four treatments: T1 - addition of commercial starters (Staphylococcus xylosus and Lactococcus lactis ssp lactis); T2 - mixture of isolated Staphylococcus xylosus more commercial Lactococcus lactis ssp lactis ; T3 - mixture of isolated Lactococcus lactis ssp lactis more commercial Staphylococcus xylosus and T4 - Staphylococcus xylosus and Lactococcus lactis ssp lactis both isolated, evaluating its influence on the microbiological, physico-chemical and sensorial characteristics. About 13.3% of the colonies isolated of sausages were identified as Staphylococcus xylosus. The maximum growth of Lactococcus lactis ssp lactis, in pork plasma culture medium was 8.58 Log UFC.mL-1 in 27 hours, and the OD 0.88. In the MRS broth the growth reached the maximum peak (8.70 Log UFC.mL-1) in 6 hours, presenting OD of 0.81. All the elaborated sausages presented a significant decreased of pH and a reduction in the water activity, ensuring a microbiological security to the products. In relation the lipid oxidation, the treatment that contained isolated strains of Staphylococcus xylosus presented significantly lower values than the other treatments. The sausages elaborated with both strains isolated presented better sensorial results than the sausages elaborated with commercial starters cultures. Therefore, this study indicated that would be promising to extend the availability of microorganisms for industrial use from the selection of native cultures, that the pork plasma culture medium becomes an alternative for the multiplication of lactic acid bacteria, because it presented a similar fermentation performance to the commercial culture medium and that the addition of natives starters cultures can be used in the elaboration of fermented Italian sausages, providing safety products and with differentiated flavor. / Este trabalho teve por objetivo produzir e testar o desempenho de culturas starters nativas na fabricação de salame tipo Italiano quanto à segurança e qualidade dos salames. No primeiro experimento, cepas de Staphylococcus xylosus foram isoladas de salames coloniais, caracterizadas e após identificadas pelo Kit Api Staph (Biomérieux). No segundo experimento, o microrganismo Lactococcus lactis ssp lactis foi fermentado em meio de cultura de plasma suíno e avaliado sua eficiência em relação ao caldo MRS, através de análises microbiológicas e densidade óptica (DO). No terceiro experimento, as culturas isoladas e multiplicadas foram adicionadas em salame, elaborando-se quatro tratamentos: T1 - adição de starters comerciais (Staphylococcus xylosus e Lactococcus lactis ssp lactis); T2 - mistura de Staphylococcus xylosus isolado mais Lactococcus lactis ssp lactis comercial; T3 - mistura de Lactococcus lactis ssp lactis isolado mais Staphylococcus xylosus comercial e T4 - Staphylococcus xylosus e Lactococcus lactis ssp lactis ambos isolados, avaliando sua influência nas características microbiológicas, físico-químicas e sensoriais. Foram identificadas 13,3% das colônias isoladas de salames coloniais como Staphylococcus xylosus. O crescimento máximo de Lactococcus lactis ssp lactis, em meio de cultura de plasma suíno, foi de 8,58 Log UFC.mL-1 no tempo de 27 horas e DO de 0,88. Em caldo MRS, o crescimento atingiu pico máximo (8,70 Log UFC.mL-1) em 6 horas, apresentando uma DO de 0,81. Todos os salames elaborados apresentaram uma queda de pH significativa e também uma redução na atividade de água, garantindo uma segurança microbiológica aos produtos. Em relação a oxidação lipídica, os tratamentos que continham cepas de Staphylococcus xylosus isolados apresentaram valores significativamente menores que os outros tratamentos. Os salames elaborados com as duas cepas isoladas (T4) apresentaram melhores resultados sensoriais quando comparados com salames elaborados com culturas starters comerciais. Portanto, este estudo indicou que seria promissor ampliar a disponibilidade de linhagens para uso industrial proveniente da seleção de culturas nativas, que o meio de cultura de plasma suíno torna-se uma alternativa para a multiplicação de bactérias ácido lácticas, pois apresentou um desempenho semelhante à fermentação do meio de cultura comercial e que a adição de culturas starters nativas pode ser utilizada na elaboração de salames, proporcionando produtos seguros e com flavor diferenciado.

L’épithélium digestif est recouvert d’une couche protectrice de mucus, qui est un hydrogel perméable et viscoélastique. La couche de mucus est formée d'un réseau de fibres de mucines. Ces dernières sont des glycoprotéines de haut poids moléculaire avec un squelette protéique riche en sérine et thréonine, lié à une grande variété de O-glycanes qui représentent une source nutritionnelle pour les bactéries et/ou des ligands potentiels pour les adhésines bactériennes, contribuant ainsi probablement à la sélection et l'implantation d'un microbiote régio-spécifique. Nous nous sommes intéressés aux capacités muco-adhésives de L. lactis TIL448 par le couplage de (i) la microscopie à force atomique (AFM), à l'échelle de la cellule unique et en mode statique et (ii) la méthode hydrodynamique en chambre à écoulement cisaillé, à l'échelle de l’ensemble de la population bactérienne. Dans l'optique d'identifier la nature et le rôle fonctionnel des déterminants de surface mis en jeu, nous avons testé, outre la souche sauvage, la souche curée de plasmides TIL1230 et deux mutants TIL1289 et TIL1290, altérés dans la synthèse de pili et d'une protéine "mucus-binding", respectivement. Pour relier les propriétés muco-adhésives et diffusives de L. lactis, les capacités de migration de la souche TIL448 et de ses dérivés ont ensuite été évaluées dans des suspensions de PGM à concentration variable (0,5% et 5% (m/v)), en mettant en œuvre une nouvelle méthode "Diffusion Front Tracking" (DFT). Cette méthode consiste à suivre le front de diffusion de la suspension bactérienne au cours du temps au sein du réseau de PGM, dans une chambre de Hele-Shaw, couplée à une caméra CCD. Les bactéries L. lactis sont préalablement marquées avec la fuschine pour mieux visualiser le front de diffusion. Par ailleurs, nous avons démontré que les bactéries L. lactis ont tendance à être plus diffusives dans PGM 0,5% (m/v) que dans PGM 5% (m/v). La microstructure du réseau de mucines a donc été caractérisée par des approches de microrhéométrie 1 point (1P) et 2 points (2P) et de suivi de particules fluorescentes / The digestive epithelium is covered with a protective mucus layer, regarded as a viscoelastic and permeable hydrogel. Mucins are large glycoproteins with a serine and threonine-rich protein backbone, linked to a wide variety of O-linked oligosaccharide side chains arranged in a bottle-brush configuration. Such O-glycans are nutritive sources for bacteria and/or potential ligands for bacterial adhesins, probably contributing in this way to the selection of the species-specific microbiota. In this thesis, we focused on unraveling multi-scale interactions between a vegetal L. lactis subsp. lactis isolate, TIL448 and a model mucin, Pig Gastric Mucin (PGM). Our study, based on the combination of different biophysical approaches and tools, has allowed dissecting the muco-adhesive and diffusive phenotype of L. lactis TIL448, in relation with the nature of the bacterial surface determinants and the structural, mechanical and rheological properties of the PGM network. Firstly, the muco-adhesion of TIL448 were examined using the single-cell scale AFM measurements with dedicated lacto-probes and shear stress flow chamber experiments at the bacterial population level, under laminar flow conditions. We also tested the plasmid-cured strain and two mutants, obtained by disruption of the genes encoding the major pilin and the mucus-binding protein. Then, the diffusion ability of L. lactis was determined by implementing a novel method, named Diffusion Front Tracking (DFT). It consists of tracking the diffusion front of stained cell suspensions over time within the PGM network. In a second part, in order to have a more thorough understanding of the L. lactis muco-adhesive and diffusive ability, the microstructure and mechanical properties of PGM were determined. Gel microstructure for varying PGM concentration was probed by the analysis of diffusivities of 200-nm and 500-nm fluorescent nanoparticles with different surface properties (carboxyl-terminated, amine-terminated and neutral charged tracers), using fluorescence Multiple-Particle Tracking

Résumé confidentiel / Résumé confidentiel