Positive role of cell wall anchored proteinase PrtP in adhesion of lactococci

<p>Abstract</p> <p>Background</p> <p>The first step in biofilm formation is bacterial attachment to solid surfaces, which is dependent on the cell surface physico-chemical properties. Cell wall anchored proteins (CWAP) are among the known adhesins that confer the adhesive properties to pathogenic Gram-positive bacteria. To investigate the role of CWAP of non-pathogen Gram-positive bacteria in the initial steps of biofilm formation, we evaluated the physico-chemical properties and adhesion to solid surfaces of <it>Lactococcus lactis</it>. To be able to grow in milk this dairy bacterium expresses a cell wall anchored proteinase PrtP for breakdown of milk caseins.</p> <p>Results</p> <p>The influence of the anchored cell wall proteinase PrtP on microbial surface physico-chemical properties, and consequently on adhesion, was evaluated using lactococci carrying different alleles of <it>prtP</it>. The presence of cell wall anchored proteinase on the surface of lactococcal cells resulted in an increased affinity to solvents with different physico-chemical properties (apolar and Lewis acid-base solvents). These properties were observed regardless of whether the PrtP variant was biologically active or not, and were not observed in strains without PrtP. Anchored PrtP displayed a significant increase in cell adhesion to solid glass and tetrafluoroethylene surfaces.</p> <p>Conclusion</p> <p>Obtained results indicate that exposure of an anchored cell wall proteinase PrtP, and not its proteolytic activity, is responsible for greater cell hydrophobicity and adhesion. The increased bacterial affinity to polar and apolar solvents indicated that exposure of PrtP on lactococcal cell surface could enhance the capacity to exchange attractive van der Waals interactions, and consequently increase their adhesion to different types of solid surfaces and solvents.</p

Surface-Related Features and Virulence Among Acinetobacter baumannii Clinical Isolates Belonging to International Clones I and II

Acinetobacter baumannii currently represents one of the most important nosocomial infection agent due to its multidrug-resistance and a propensity for the epidemic spread. The A. baumannii strains belonging to the international clonal lineages I (IC I) and II (IC II) are associated with the hospital outbreaks and a high virulence. However, the intra and inter lineage-specific features of strains belonging to these most worldwide spread A. baumannii clones are not thoroughly explored. In this study we have investigated a set of cell surface-related features of A. baumannii IC I (n = 20) and IC II (n = 16) lineage strains, representing 30 distinct pulsed-field gel electrophoresis types in the collection of clinical isolates obtained in Lithuanian tertiary care hospitals. We show that A. baumannii IC II strains are non-motile, do not form pellicle and display distinct capsular polysaccharide profile compared with the IC I strains. Moreover, in contrast to the overall highly hydrophobic IC I strains, IC II strains showed a greater variation in cell surface hydrophobicity. Within the IC II lineage, hydrophilic strains demonstrated reduced ability to form biofilm and adhere to the abiotic surfaces, also possessed twofold thicker cell wall and exhibited higher resistance to desiccation. Furthermore, these strains showed increased adherence to the lung epithelial cells and were more virulent in nematode and mouse infection model compared with the hydrophobic IC II strains. According to the polymerase chain reaction-based locus-typing, the reduction in hydrophobicity of IC II strains was not capsule or lipooligosaccharide locus type-dependent. Hence, this study shows that the most widespread A. baumannii clonal lineages I and II markedly differ in the series of cell surface-related phenotypes including the considerable phenotypic diversification of IC II strains at the intra-lineage level. These findings suggest that the genotypically related A. baumannii strains might evolve the features which could provide an advantage at the specific conditions outside or within the host

Caractérisation génétique de facteurs moléculaires de l'enveloppe bactérienne associés à la croissance immobilisée des bactéries

Dans le cadre de l étude des facteurs impliqués dans l immobilisation des bactéries,nous nous sommes intéressés à deux polymères de la paroi, cruciaux pour la formation des biofilms : les exopolysaccharides (EPS) et le peptidoglycane (PG).Nous avons étudié le rôle des EPS de la capsule (CPS) dans la croissance immobilisée de Streptococcus pneumoniae en utilisant un milieu modèle semi-liquide. Nos résultats (i) montrent que des mutants CPS- s échappent des colonies immobilisées, (ii) suggèrent un mécanisme de variation de phase impliquant le gène tts de la synthèse de la CPS et (iii) indiquent l existence d une pression de sélection contre l immobilisation.Puisque l intégrité du PG affecte la formation des biofilms, nous avons cherché de nouveaux facteurs moléculaires impliqués dans sa synthèse ou sa dégradation en utilisant la bactérie modèle Lactococcus lactis. L un de ces facteurs est le gène aslA, responsable de l incorporation du D-Aspartate dans le PG, que nous avons identifié grâce à une analyse soustractive de génome. L inactivation conditionnelle de ce gène nous a permis de confirmer son rôle prédit et de montrer qu il est essentiel. Nous avons également cherché les gènes permettant de réguler l activité lytique des hydrolases du PG. Parmi eux, nous avons identifié les gènes codant pour une dé-N-acétylase (pgdA) et une O-acétylase (oatA) du PG ainsi qu un régulateur de oatA : SpxB. Nos résultats nous ont permis de caractériser la cascade de régulations qui permet à la cellule, à partir d un signal transduit par le système à deux composantes CesSR, de contrer l hydrolyse de son PG en le modifiant par une O-acétylation accrue.We studied two cell wall polymers implicated in biofilm formation: exopolysaccharide (EPS) and peptidoglycan (PG). Our investigation of capsule exopolysaccharides (CPS) of Streptococcus pneumoniae revealed that (i) CPS- mutants emerge from immobilized colonies in semi-liquid medium, (ii) a phase variation mecanism may be implied in appearance of mutations in tts gene, responsible for CPS synthesis, (iii) immobilisation is the factor providing a selective pressure. Since PG integrity affects biofilm formation, we looked for new molecular factors affecting PG synthesis or degradation. One of these factors, identified by subtractive genome analysis, is the gene aslA, responsible for D-Asp incorporation in the PG. Its conditional inactivation allowed us to confirm its function as D-Asp ligase and to show that it is indispensable. We also identified genes which could regulate lytic activity of PG hydrolysis and in thus affect PG integrity. We found three genes, overexpression if which leads to more resistant to hydrolysis PG: pgdA , oatA and spxB, encoding respectively for a PG de-acetylase, a PG O-acetylase, and the positive regulator of oatA. Our results allowed us to reveal the multi-step cascade of regulatory mechanisms, provoked by cell wall stress. It starts with induction of two component system CesSR in response to cell wall stress, and ends up with O-acetylation of PG by OatA as a mean of rendering it more resistant to hydrolytic damage. SpxB appeared to be a missing link between response to cell envelope stress and PG modification.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Dynamique de formation des biofilms de Lactococcus lactis et réactivité vis-à-vis de Listeria monocytogenes

La qualité hygiénique des aliments est une préoccupation constante des industriels de l agroalimentaire et des consommateurs. La présence de biofilms microbiens peut engendrer des résistances à l action d agents antimicrobiens, entraîner une contamination des denrées alimentaires en cours de fabrication, et lorsque les microorganismes impliqués sont pathogènes comme dans le cas de Listeria, avoir une incidence particulièrement importante sur le plan de la santé publique. Ces biofilms peuvent dans certains cas être constitués de flores technologiques positives et empêcher l implantation de flores négative. C est le principe de l écologie microbienne dirigée. Nous avons utilisé pour cela le modèle d interaction Lactococcus lactis Listeria monocytogenes, afin de mieux comprendre les phénomènes impliqués lorsqu une bactérie pathogène arrive en contact avec une surface déjà colonisée par un biofilm résident. Les résultats obtenus ont permis de mettre en évidence certains mécanismes clefs à l origine d une inhibition d implantation de Listera monocytogenes sur une surface d un biofilm résident. Nous avons en particulier démontré l effet antiadhésif vis-à-vis du pathogène de polysaccharides produits par certaines souches de Lactococcus lactis en biofilm. Les résultats de cette thèse contribueront à une meilleure gestion du risque Listeria sur les surfaces des ateliers alimentaires, en identifiant des situations à risques (biofilms résidents rugueux ou poreux) ou en proposant des critères de sélection de biofilms positifs interférant avec l implantation du pathogène (nature de la matrice, inhibition physiologique ).Food hygiene is a constant preoccupation in the food industry as well as for consumers. The presence of pathogens biofilms i.e. Listeria monocytogenes generate resistance against antimicrobial agents, leading to the contamination of food during processing, and thus pose a dangerous risk to public health. The main aim of this thesis project was to investigate the phenomena surrounding the events in which a pathogenic bacterium comes into contact with a resident biofilm, using existing Lactococcus lactis-Listeria monocytogenes interaction models. The obtained results allowed us to identify key biofilm factors that influence Listeria s initial interaction with the resident biofilm, in particular the anti-adhesive effect of synthesized exopolyssaccharides against Listeria s settlement. Moreover, the study of L. monocytogenes dynamic development in mono-species or multi-species biofilm demonstrated the inhibitory effect by the presence of lactic acid bacteria, which led to a better understanding of Lactococci-Listeria interaction as well as the spatial development of Listeria cells inside mixed community biofilms. In conclusion, the results produced during this thesis will help contribute to the improvement of risk assessment of processing surfaces in the food industry, by identifying hazardous situations (rough or porous resident biofilms) or by proposing a selection of positive biofilms criteria (matrix composition, physiological inhibition) against pathogenic settlement and colonization.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Réponse des lactocoques aux endommagements de la paroi

Le peptidoglycane (PG) est le composant essentiel et majoritaire de l'enveloppe bactérienne. Sa fonction principale est de préserver l'intégrité de la cellule contre la pression osmotique interne. L'apparition des coupures du PG par des autolysines fait partie du processus normal de la croissance de la bactérie, du renouvellement de la paroi cellulaire, de la séparation des cellules, de la germination des spores, de l'autolyse et de la formation des biofilms. Dans la présente étude concernant la réponse des lactocoques à l'activité des hydrolases du PG dans leur paroi, nous avons montré que la résistance à l'hydrolyse par le lysozyme de la bactérie à Gram positif, non pathogène Lactococcus lactis dépend du degré d'acétylation du PG effectué par la surexpression de la déacétylase, PgdA, ou de l'O-acétylase, OatA. Également, nous avons identifié le gène spxB comme un possible régulateur positif d'oatA, et nous avons démontré que son expression est sous le contrôle du système régulateur à deux composants CesSR, lequel répond au stress subit par l'enveloppe cellulaire. Dans notre travail nous avons montré que les gènes qui participent à la réponse au stress de l'enveloppe cellulaire sont également responsables, non seulement de la résistance au lysozyme, mais aussi de la résistance aux antibiotiques aminoglycosidiques et à la vancomycine.The peptidoglycane (PG) is the essential component of the bacterial cell wall. Its principal function is to preserve the integrity of the cell from internai osmotic pression. The introduction of breaks by PG 1 hydrolases is part of normal bacterial cell wall growth, processus and is involved in spore germination, autolysis and biofilm formation. ln the present study of response to cell wall damage by PG hydrolysis, we show that resistance to lysozyme of Gram positive non pathogen bacteria Lactococcus lactis depends on the degree deacetylation of PG : the overexpression of PG déacétylase PgdA or O-acétylase OatA both conferred resistance to the hydrolysis. Also we identified the gene spxB as a possible positive regulator of oatA, and showed that its expression is under the control of the two components system CesSR, which responds to cell wall stress. We showed that genes which take part in the response to the cell wall stress are responsible not only for lactococcal resistance to the lysozyme but also for resistance to aminoglycosidic antibiotics and the vancomycine.COMPIEGNE-BU (601592101) / SudocSudocFranceF

Spatial competition with Lactococcus lactis in mixed-species continuous-flow biofilms inhibits Listeria monocytogenes growth

Surfaces in industrial settings provide a home for resident biofilms that are likely to interact with the attachment, growth and survival of pathogens such as Listeria monocytogenes. Experimental results have indicated that L. monocytogenes cells were inhibited by the presence of a model resident flora (Lactococcus lactis) in dual-species continuous flow-biofilms, and are spatially restricted to the lower biofilm layers. Using a new, simplified individualbased model (IBM) that simulates bacterial cell growth in a three-dimensional space, the spatial arrangements of the two species were reconstructed and their cell counts successfully predicted. This model showed that the difference in generation times between L. monocytogenes and L. lactis cells during the initial stages of dual-species biofilm formation was probably responsible for the species spatialization observed and the subsequent inhibition of growth of the pathogen. © 2011 Taylor & Francis.Link_to_subscribed_fulltex

Genetic Features of Resident Biofilms Determine Attachment of Listeria monocytogenes▿

Planktonic Listeria monocytogenes cells in food-processing environments tend most frequently to adhere to solid surfaces. Under these conditions, they are likely to encounter resident biofilms rather than a raw solid surface. Although metabolic interactions between L. monocytogenes and resident microflora have been widely studied, little is known about the biofilm properties that influence the initial fixation of L. monocytogenes to the biofilm interface. To study these properties, we created a set of model resident Lactococcus lactis biofilms with various architectures, types of matrices, and individual cell surface properties. This was achieved using cell wall mutants that affect bacterial chain formation, exopolysaccharide (EPS) synthesis and surface hydrophobicity. The dynamics of the formation of these biofilm structures were analyzed in flow cell chambers using in situ time course confocal laser scanning microscopy imaging. All the L. lactis biofilms tested reduced the initial immobilization of L. monocytogenes compared to the glass substratum of the flow cell. Significant differences were seen in L. monocytogenes settlement as a function of the genetic background of resident lactococcal biofilm cells. In particular, biofilms of the L. lactis chain-forming mutant resulted in a marked increase in L. monocytogenes settlement, while biofilms of the EPS-secreting mutant efficiently prevented pathogen fixation. These results offer new insights into the role of resident biofilms in governing the settlement of pathogens on food chain surfaces and could be of relevance in the field of food safety controls