Study of the molecular bases governing biofilm formation in Bacillus cereus

Conclusiones: Entre las conclusiones obtenidas, cabe destacar: - B. cereus produce fibras tipo amiloides necesarias para el correcto desarrollo estructural del biofilm. - El exopolisacárido denominado EPS1 está implicado en la movilidad tipo swarming de la colonia, mientras que el EPS2 tiene un papel determinante en la adhesión y en la formación de biofilm. Ambos polisacáridos tienen un papel sinérgico en la protección de la colonia. - Los individuos que forman parte del biofilm sobrexpresan los elementos relacionados con la defensa frente al ataque del huésped o competidores, mientras que las células en estado planctónico muestran un estado de ataque, sobrexpresando toxinas y elementos para un ataque rápido y efectivo. Fecha de lectura de Tesis: 6 de julio 2018.Introducción o motivación de la tesis: B. cereus es un patógeno humano responsable de múltiples enfermedades, destacando por ser más comunes las intoxicaciones alimentarias por el consumo de alimentos contaminados en origen. La formación de biofilms está directamente relacionada con la contaminación de alimentos, bien por favorecer la adhesión de las bacterias sobre la superficie de los alimentos o como reservorio de esporas y células vegetativas en la industria alimentaria, contaminando lotes completos de productos comerciales. El estudio de las bases moleculares de la formación de biofilm en esta bacteria es escaso, lo que ha motivado la realización de esta tesis doctoral. Desarrollo teórico y metodología: Para el desarrollo de la tesis doctoral se ha utilizado un triple enfoque genético, transcriptómico y proteómico. Se ha estudiado la formación de fibras de proteínas tipo amiloides como elemento estructural indispensable para la formación de biofilm y se han caracterizado bioquímicamente dos proteínas implicadas directamente en la formación de estas fibras. De forma general, los exopolisacáridos también constituyen un elemento principal en la formación de biofilm. Se han estudiado dos regiones génicas implicadas en la síntesis de exopolisacáridos, siendo uno de ellos imprescindible para el correcto desarrollo del biofilm. Además, el enfoque transcriptómico y proteómico ha permitido la identificación de múltiples funciones y adaptaciones de B. cereus al pasar al estilo de vida sésil y en comunidad que supone la formación de un biofilm

Plant polyphenols inhibit functional amyloid and biofilm formation in <i>Pseudomonas</i> strains by directing monomers to off-pathway oligomers

Self-assembly of proteins to &beta;-sheet rich amyloid fibrils is commonly observed in various neurodegenerative diseases. However, amyloid also occurs in the extracellular matrix of bacterial biofilm, which protects bacteria from environmental stress and antibiotics. Many Pseudomonas strains produce functional amyloid where the main component is the highly fibrillation-prone protein FapC. FapC fibrillation may be inhibited by small molecules such as plant polyphenols, which are already known to inhibit formation of pathogenic amyloid, but the mechanism and biological impact of inhibition is unclear. Here, we elucidate how polyphenols modify the self-assembly of functional amyloid, with particular focus on epigallocatechin gallate (EGCG), penta-O-galloyl-&beta;-d-glucose (PGG), baicalein, oleuropein, and procyanidin B2. We find EGCG and PGG to be the best inhibitors. These compounds inhibit amyloid formation by redirecting the aggregation of FapC monomers into oligomeric species, which according to small-angle X-ray scattering (SAXS) measurements organize into core-shell complexes of short axis diameters 25&ndash;26 nm consisting of ~7 monomers. Using peptide arrays, we identify EGCG-binding sites in FapC&rsquo;s linker regions, C and N-terminal parts, and high amyloidogenic sequences located in the R2 and R3 repeats. We correlate our biophysical observations to biological impact by demonstrating that the extent of amyloid inhibition by the different inhibitors correlated with their ability to reduce biofilm, highlighting the potential of anti-amyloid polyphenols as therapeutic agents against biofilm infections

Overcoming biofilm tolerance by a novel approach targeting bacterial persisters

Multi-drug tolerance is a phenomenon, in which microorganisms normally susceptible to an antimicrobial agent are able to withstand a treatment via phenotypic alteration. The tolerance is conveyed by a microbial subpopulation that is in a non-replicative and metabolically inactive state also known as persistence. Through this kind of dormancy, the subpopulation may survive an otherwise appropriate course of antimicrobials, since the majority of the drugs target cellular division or metabolism. Upon the reduction of the surrounding antimicrobial concentration the multi-drug tolerant cells - persisters - become resuscitated thus allowing repopulation. As opposed to the more widely acknowledged challenge of antimicrobial resistance, the offspring of the specialist survivor cells are genetically identical to the susceptible majority. Persisters are especially abundant in biofilms, a microbial lifestyle characterized by aggregated microcolonies that are covered in a self-produced slimy matrix known as extracellular polymeric substance (EPS). Partly owning to this protective matrix, biofilms are inherently somewhat tolerant to antimicrobial chemotherapy. Moreover, microbes confined in a biofilm are additionally protected against the components of the host immune system. Conversely, it is assumed that persisters in planktonic, i.e. freely floating state, are easily cleared out by white blood cells. Combined, the immune evasive properties of biofilms and the remarkable multi-drug tolerance of persisters give rise to recalcitrant infections that are immensely difficult to eradicate. The described phenomenon constitutes crucially to the major healthcare challenge of chronic, treatment-resistant infections. Tuberculosis, cystic fibrosis lung disorder, bacterial endocarditis and infections related to indwelling medical devices are only a few examples of such problems. Despite the need for antimicrobials with anti-persister efficacy, no such therapeutics is available and very few are being investigated - one important factor being the lack of relevant drug discovery platforms. Therefore, the aim of this study was to develop an anti-persister assay and to carry out a pilot screening of natural product derived bioactive compounds. Based on the notion that persisters are enriched in bacterial cultures that have reached the stationary phase of growth, a persister model was designed using Staphylococcus aureus ATCC 25923 as the test strain. The bacteria were grown in liquid cultures until they reached the stationary phase and subsequent experimentation was carried out to confirm the tolerant state. After the stationary phase persister model was validated, a small pilot screening of natural products was undertaken in the hope of finding novel anti-persister activity. Mitomycin C, a cytotoxic drug with an existing anti-cancer indication was assigned as the positive control compound because of its previously established anti-persister activity. Since it is common for all of the persister-related diseases that the target microorganisms reside within a protective biofilm, an additional assay based on biofilm regrowth was designed to characterize the hit compounds on a more clinically relevant platform. The persister model culture was shown to be tolerant to conventional antibiotics. The re-induction of metabolic activity by diluting into fresh medium recovered the antimicrobial susceptibility expectedly. A total of 4 compounds were identified as anti-persister hits in the pilot screening campaign. Chromomycin A3, dehydroabietic acid, mithramycin A and oleanolic acid were all able to reduce the viable bacterial count in the stationary phase persister model more than 2 logarithmic units at 100 µM. Mithramycin A was the most potent, reducing the viability over 6 log units. The model compound mitomycin C reduced the viable counts 5.49 (± 0.96) logarithmic units. Out of the 4 hits, dehydroabietic acid was selected for the biofilm relapse assay because of its favourable biocompatibility properties. It reduced regrowth for the treated biofilms by 4 logarithmic.Monilääketoleranssi (multi-drug tolerance) on ilmiö, jossa antimikrobilääkkeelle herkät mikro-organismit kykenevät sietämään lääkehoitoa fenotyyppiään muokkaamalla. Toleranssin aikaansaa jakaantumattomassa ja metabolisesti epäaktiivisessa tilassa elävä osapopulaatio. Tämänkaltaisesta horrostilan tuomasta sietokyvystä käytetään nimitystä persistenssi. Sietokykyinen osapopulaatio voi selviytyä asianmukaisesti valitusta antimikrobilääkehoidosta, sillä lääkkeiden vaikutus kohdistuu tyypillisesti juuri solunjakaantumistoimintoihin tai aineenvaihduntaan. Kun ympäröivä lääkeainepitoisuus kuurin päätyttyä laskee, nämä monilääketolerantit solut - persisterit - elpyvät uudelleen kansoittaakseen tuhoutuneen populaation. Toisin kuin paremmin tunnetussa antimikrobilääkeresistenssin ilmiössä, näiden erikoistuneiden selviytyjäsolujen jälkeläiset ovat geneettisesti identtisiä lääkkeelle herkän osapopulaation kanssa. Persisterisoluja on erityisen runsaasti biofilmeissä. Biofilmi on yhteen takertuneiden bakteerien muodostama yhdyskunta, joka tuottaa ympärilleen tyypillisen, solunulkoisten polymeerien muodostaman limakerroksen (extracellular polymeric substance, EPS). Osittain tämän suojaavan liman ansiosta biofilmit ovat itsessään melko sietokykyisiä antimikrobilääkkeille. Lisäksi biofilmissä elävät mikrobit ovat erityisen hyvin suojattuja isäntäorganismin immuunijärjestelmää vastaan. Sen sijaan planktoniset, yksittäisinä soluina irrallaan kelluvat mikrobisolut ovat helposti valkosolujen hävitettävissä. Biofilmin immunoevasiiviset ominaisuudet yhdistettynä persisterien monilääketoleranssiin mahdollistavatkin erittäin vaikeahoitoisten, kroonisten infektioiden kehittymisen. Kuvattu sietokykymekanismi kroonistuneiden, hoitoon vastaamattomien infektioiden taustalla muodostaa merkittävän terveysuhan. Tuberkuloosi, kystisen fibroosin keuhko-ongelmat, bakteeriendokardiitti ja lääkinnällisiin laitteisiin liittyvät infektiot ovat joitakin esimerkkejä tällaisista hoidollisista haasteista. Erityisesti persistereihin tepsivien antimikrobilääkkeiden huutavasta tarpeesta huolimatta yhtäkään ei ole käytettävissä ja vain harvoja tutkitaan. Soveltuvien lääkekehitysalustojen puute on yksi olennainen syy tähän uusien keksintöjen puutteeseen. Sen vuoksi tämän työn tarkoituksena onkin kehittää menetelmä persisterilääkkeiden tunnistamiseksi ja tehdä pienen mittakaavan pilottiseulonta luonnontuotteista eristetyille, bioaktiivisille yhdisteille. Persisterien on havaittu rikastuvan stationäärivaiheen bakteeriviljelmissä. Sen vuoksi persisterimallia ryhdyttiin kehittämään Staphylococcus aureus ATCC 25923 -bakteerikannalla, jota kasvatettiin nesteviljelminä staattiseen kasvuvaiheeseen saakka. Malliviljelmän lääketoleranssi tutkittiin, minkä jälkeen se altistettiin valikoiduille luonnontuotteille tarkoituksena löytää persisteribakteereihin tehoavia yhdisteitä. Positiiviseksi kontrolliksi valittiin mitomysiini C - syöpälääkkeenä käytettävä sytotoksinen yhdiste, jonka on aiemmin todettu tuhoavan myös persisteribakteereita. Koska kliinisesti merkittävät persisteri-ilmentymät tavataan juuri biofilmeissä, päätettiin lisäksi kehittää kasvun uudelleenkäynnistymistä biofilmistä mittaava menetelmä seulontalöytöjen tarkempaan kartoittamiseen hoidollisesti relevanteissa olosuhteissa. Persisterimalli havaittiin sietokykyiseksi perinteisille antibiooteille. Metabolisen aktiivisuuden uudelleenkäynnistäminen tuoreeseen ravinneliuokseen laimentamalla palautti lääkeherkkyyden odotetusti. Seulonta löysi yhteensä 4 persistereihin tehoavaa yhdistettä: kromomysiini A3, dehydroabietiinihappo, mitramysiini A ja oleanolihappo kykenivät kaikki laskemaan elävien bakteerien lukumäärää persisterimallissa yli 2 logaritmiyksikköä 100 µM pitoisuuksina. Mitramysiini A oli yhdisteistä tehokkain vähentäen bakteerilukua yli 6 yksikköä. Malliyhdiste mitomysiini C vähensi elävien bakteerien lukua vastaavasti 5.49 (± 0.96) logaritmiyksikköä. Dehydroabietiinihappo valittiin jatkotutkimukseen biofilmin uudelleenkasvukykykokeeseen sen suosiollisen toksisuusprofiilin perusteella. Se vähensi uudelleenkasvua hoidetusta biofilmistä 4 logaritmiyksikköä osoittaen siten merkittävää aktiivisuutta

Screening of aerobic endospore-forming bacterial isolates as candidate biocontrol agents against rhizoctonia solani.

Doctor of Philosophy in Microbiology. University of KwaZulu-Natal, Pietermaritzburg 2016.Bacterial-based biocontrol of soil-borne phytopathogens has gained prominence as a promising technology for developing sustainable agricultural pest control practices. Aerobic endospore-forming bacteria are seen as potential candidates for biocontrol applications due to various ecological and physiological traits which have been shown to influence plant health and disease control. Their ability to produce endospores also provides a major commercial advantage over non spore-forming bacteria. Appropriate screening methods are central to the discovery of successful biocontrol agents and should ideally be both ecologically relevant and able to evaluate a large number of isolates. A study was therefore undertaken with the aim of establishing screening methods that facilitate the selection of aerobic endospore-forming bacteria as candidate biocontrol agents against Rhizoctonia solani, an economically important fungal pathogen exhibiting a wide host range. Aerobic endospore-forming bacteria were isolated from rhizosphere material of five crop types grown in composted pine bark medium and screened for R. solani antagonism using traditional in vitro dual-culture bioassays. Isolates exhibiting antifungal activity were then evaluated in vivo for biocontrol activity against R. solani in cucumber seedling trials. Selected isolates were evaluated further using several screening approaches including: genomic fingerprinting; characterization of, and PCR-based screening for genes involved in the biosynthesis of bioactive lipopeptide compounds; and, the use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) as a means of rapidly screening bacterial isolates. Approximately 6% of the bacterial isolates (n=400) showed antagonism towards R. solani in vitro. Dual-culture bioassays against R. solani, Fusarium oxysporum, Botrytis cinerea and Pythium arrhenomanes revealed that the antagonistic activity amongst isolates varied considerably and was influenced by the duration of the assay. From these assays it was possible to rank isolates based on the extent and stability of the inhibitory response in vitro as well as by the spectrum of antifungal activity observed. Twenty-four isolates were selected for in vivo screening for biocontrol activity against R. solani, using susceptible cucumber plants grown under greenhouse conditions. In preliminary experiments the pathogen loading rates were shown to have a marked influence on disease severity. In experiments where R. solani was seeded in the form of colonized agar plugs, significant differences between treatments and controls were recorded and several potential biocontrol candidates were identified. A general observation was that isolates that achieved high rankings in vitro performed better in the in vivo trial than those with lesser rankings; although some exceptions were noted. These findings support the notion that fungal antagonism is an important determinant of biocontrol potential that can be used in preliminary biocontrol screening programmes. Internal-transcribed spacer region (ITS) PCR and randomly amplified polymorphic DNA (RAPD) PCR were evaluated as methods to differentiate isolates exhibiting antifungal activity in vitro. ITS-PCR distinguished three major groupings, but proved to be limited in its ability to detect inter- and intra-specific variation amongst closely related organisms. Based on 16S rRNA gene sequence analysis, two of the groups were identified as members of the “Bacillus subtilis” and “Bacillus cereus” clusters; while, the third group consisted of a single isolate identified as a strain of Brevibacillus laterosporus. RAPD-PCR revealed further levels of genetic diversity within each ITS grouping. The “Bacillus subtilis” cluster was distinguished further into four distinct groups, which based on gyrA gene fragment sequence analysis, were identified as strains of B. amyloliquefaciens subsp. plantarum and B. subtilis respectively. Sequence matches were consistent with the RAPD-PCR groupings, indicating that this method was appropriate for differentiating related isolates at the strain and possibly the sub-species level. Clonal similarities were evident for a number of strains isolated from different plant species suggesting that these may reflect populations of rhizosphere competent strains and/or plant adapted ecotypes. Strains of B. amyloliquefaciens subsp. plantarum and B. subtilis were amongst the best performers in the in vivo biocontrol seedling trial and generally performed better than the “Bacillus cereus” group of isolates. RAPD-PCR of the “Bacillus cereus” isolates showed that they exhibited greater levels of genetic heterogeneity and that the groupings detected were not consistent when different primer sets were evaluated. Genomic fingerprinting was found to provide an insight into the prevalence, distribution and possible rhizosphere competency of related strains. Liquid chromatography was used in conjunction with electrospray-ionization time-of-flight (ESI-TOF) mass spectrometry (MS) to characterize bioactive lipopeptides purified from culture supernatants of selected strains that ranked highly in the in vitro/in vivo assays. Phylogenetically related strains produced very similar lipopeptide profiles. Bacillus subtilis strains were found to produce isoforms of surfactin and fengycin. In addition to these lipopeptides, B. amyloliquefaciens subsp. plantarum strains were also found to produce isoforms of bacillomycin D or iturin A. Bacillomycin/iturin and fengycin fractions exhibited antifungal activity in vitro, whereas surfactin fractions did not. Isolates that ranked the highest in the R. solani dual-culture bioassays all produced either isoforms of bacillomycin D or iturin A. Bacillomycin D producing isolates were amongst the best performers in the in vivo biocontrol trials. Gene markers targeting the biosynthetic apparatus of the detected lipopeptide classes were then assessed for screening purposes using PCR. BACC1F/1R primers targeting the bacillomycin D synthetase C (bmyC) gene correlated well with the ESI-TOF MS findings, whereas ITUD1F/1R primers targeting the malonyl-CoA-transacylase (ituD) gene linked to iturin A biosynthesis were unable to distinguish between isolates that produced iturin or bacillomycin in culture. Disparities between some of the PCR and ESI-TOF MS results suggested that primers targeting srfA (surfactin) and fenD (fengycin) biosynthetic genes showed limited specificity amongst the strains screened. Phylogenetic comparisons of srfD and fenD gene sequences from selected strains of B. amyloliquefaciens subsp. plantarum and B. subtilis revealed that these genes clustered according to species with marked heterogeneity between clusters being evident. Using fenD gene sequence data from B. amyloliquefaciens subsp. plantarum FZB42, primers (FENG1F/1R) targeting fengycin synthetase genes of strains of B. amyloliquefaciens subsp. plantarum isolated in this study were successfully established. MALDI-TOF MS was assessed as a means of identifying isolates antagonistic to R. solani in vitro and determining their associated lipopeptide profiles. Mass spectra were obtained in the m/z range 2000 to 20000 for identification and grouping purposes and in the m/z 750 to 2500 range in order to profile lipopeptide production. The available Bruker BDal spectral library allowed for the identification of isolates to the genus level but proved to be limited for identifying environmental isolates to the species level. Extension of the library using “inhouse” mass spectra generated from isolates identified in this study significantly improved the level of isolate identification in subsequent identification runs. Cluster analysis of mass spectra allowed for the relationships between isolates to be established and provided a means of grouping closely related isolates. Strains of B. subtilis and B. amyloliquefaciens were clearly distinguished from one another and the potential for differentiating strains at the subspecies level was also shown. MALDI-TOF MS also provided a convenient means of detecting bioactive lipopeptides directly from whole cell preparations, cell extracts and crude culture filtrates. Lipopeptide profiles varied depending on taxonomic groupings. Results for isolates within the “Bacillus subtilis” group supported the earlier ESI-TOF MS findings and were found to be more reliable than PCR screening for lipopeptide synthesis genes. “Bacillus cereus” group isolates produced distinct spectral profiles with peaks that were consistent with biomarkers previously described in the literature as isoforms of the kurstakin class of lipoheptapeptides. Brevibacillus laterosporus CC-R4 yielded a unique spectral profile in the m/z 750-2000 range with mass fragments which were similar to antimicrobial compounds recently reported in the literature. Overall, MALDI-TOF MS was found to fulfil the requirement for a practical yet robust technique suitable for processing large numbers of aerobic endospore-forming bacteria for biocontrol screening. This study has shown that genomic fingerprinting and MALDI-TOF MS characterization of bacterial isolates are worthwhile additions to preliminary in vitro screening practices. They provide a level of isolate differentiation and characterization that is beneficial for selecting candidate biocontrol agents, which is not possible with traditional screening practices. Effectively, they allow traditional biocontrol screening to move away from empirically based approaches to ones which are “knowledge” based, allowing for representative groups of bacteria with specific traits to be selected for further evaluation

Substances exopolymériques de biofilms bactériens : quantification in situ et étude de leur rôle dans la cohésion de la matrice extracellulaire

Les biofilms représentent une contrainte technologique dans le secteur industriel et sont à l'origine de nombreux cas d'infections chroniques dans le domaine médical. De ce fait, la compréhension des processus biologiques qui s'opèrent au sein de ces communautés microbiennes est un défi permanent. Des méthodes spécifiques, sensibles et diversifiées s'avèrent essentielles pour apporter une connaissance approfondie de l'organisation des biofilms en réponse à des facteurs environnementaux. La matrice extracellulaire qui englobe les microorganismes constitue un réseau complexe, et la description de sa composition et de sa structure constitue un enjeu majeur. Des outils de caractérisation in situ et non-destructifs des Substances ExoPolymériques (SEP) ont pu être proposés lors de ce travail de thèse : des méthodes de quantification ciblant spécifiquement chaque composant majeur de la matrice extracellulaire ont ainsi été développées et validées sur des biofilms modèles. Ces biofilms, choisis pour leur diversité en terme de matrice extracellulaire sont formés par les souches Pseudomonas aeruginosa ATCC 15442, Bacillus licheniformis CIP 110824 et Weissella confusa LBAE-UPS C39-2. Des critères communs ont été retenus pour guider le choix des marqueurs retenus pour développer les dosages : spécificité pour la détection de toutes les formes moléculaires d'une même famille biochimique, sensibilité pour la détection de faibles quantités de polymères dans des biofilms en microplaque, et possibilité d'observation en microscopie basée sur la détection de fluorescence. Une stratégie commune a été adoptée pour la quantification des exoprotéines (ePN), exopolysaccharides (ePS) et fibres amyloïdes (FA) de la matrice et a consisté à : (i) définir une molécule standard pour calibrer le test ; (ii) analyser d'éventuelles interférences en solution ; (iii) valider la faisabilité et la fiabilité du test in situ par la méthode des ajouts dosées réalisée sur chacun des biofilms modèles. Un composé naturel pro-fluorescent, l'épicocconone, a été retenu pour la quantification des exoprotéines. Le test a montré une limite de détection de 0,2 µg par puits, sans aucune interférence significative des autres composants majeurs du biofilms (ePS, ADNe, cellules). D'autre part, les protéines sous forme amyloïdes ont pu être détectées avec la même sensibilité que les non amyloïdes par ce marqueur. Par la suite, les exopolysaccharides ont été dosés en exploitant la réaction de Schiff, et la méthode a permis d'obtenir une limite de détection de 0,3 µg par puits. Les protéines amyloïdes bactériennes (FA) ont également été quantifiées au moyen du marqueur Thioflavine T. La k-caséine a été utilisée comme étalon, après fibrillation de la protéine native in vitro. Deux types de dosages ont été développés : le dosage ex situ de FA extraites de biofilms bactériens et le dosage in situ des protéines amyloïdes au cœur des biofilms. Enfin l'ADN extracellulaire (ADNe) a également été quantifié à partir du PicoGreen(r) tout en contrôlant le temps d'exposition du marqueur avec les biofilms afin de ne marquer que l'ADN extracellulaire. Un profil quantitatif des SEP a ainsi pu être établi pour les trois souches modèles. Le second objectif du travail s'est focalisé sur la compréhension du rôle des SEP dans les propriétés d'adhérence et de cohésion du biofilm de Bacillus licheniformis, responsable de la colonisation de surfaces dans l'industrie papetière. Les outils ainsi développés ont permis d'étudier la contribution de chaque composé matriciel (ePN, ePS, FA, ADNe) dans le comportement du biofilm, en réponse à des actions enzymatiques. La combinaison de ces dosages à des observations en microscopie confocale et à des mesures de perméabilité du biofilm a alors permis de proposer une interaction possible entre les fibres amyloïdes et l'ADN extracellulaire. Ce complexe stable pourrait agir comme facteur d'adhérence et d'agrégation intercellulaire pour assurer la stabilité et la structure tridimensionnelle du biofilm.Biofilms are detrimental in many industrial and medical areas and understanding of biofilms processes has been a challenge for decades. Specific, sensitive and rapid methods for monitoring biofilm formation are essential for a deep knowledge of biofilm organization and response to environmental factors. In such complex network that constitutes the biofilm matrix, it has become a major issue to succeed in describing its composition and local structure to determine the interactions that govern the biofilm formation. Non-destructive and in situ methods for Exopolymeric Substances (EPS) characterization were proposed along this PhD work. The first aim of the study was to propose some quantitative tools that would specifically target each major compound of the biofilm matrix. Some performance criteria were commonly established to guide the choice of each dye and to validate each step of the analytical development. These requirements can be displayed in terms of biochemical specificity, sensitivity and applicability on fluorescence-based microscopy. A common experimental strategy was carried out to quantify the exoproteins (ePN), exopolysaccharides (ePS) and amyloid fibrils (AF) and aimed at (i) choosing a calibration standard; (ii) analyzing in vitro interferences; (iii) validating the practicability and the reliability of each proposed method for an in situ implementation on biofilms. This last criteria was verified by implementing the Standard Addition Method (SAM). For that purpose, a first method was developed to take advantage of a natural pro-fluorescent dye, called epicocconone, to quantify the ePN of three model bacterial biofilms formed by the strains Bacillus licheniformis CIP 110824, Pseudomonas aeruginosa ATCC 15442 and Weissella confusa LBAE-UPS C39.2. The three bacterial models were chosen because of their contrasted EPS matrix composition. This method showed a detection limit of about 0.2µg per well and no significant interference were revealed. Moreover the epicocconone assay was able to quantify the AF with the same sensitivity as the other proteins. Subsequently, exopolysaccharides from the same strains were assayed by applying the Periodic acid-Schiff reaction in a microplate format. This chemical reaction targets the majority of the hydroxyl groups of carbohydrate and allows to give an exhaustive estimation of the sugar content of the matrix with a detection limit of 0.3µg per well. Bacterial amyloids were also specifically quantified by the using of the benzothiazole dye Thioflavine T (ThT). An amyloidogenic protein, the ?-casein, was used as standard after an in vitro fibrillation of the native form. Two types of assay were performed with, firstly, an ex situ quantification of the extracted AF and secondly, an in situ assay directly implemented on intact biofilms. The eDNA was assayed by using the PicoGreen(r) and the dye exposure-time was monitored to ensure the specificity for the extracellular DNA. These tools led to establish a quantitative EPS profile of each model biofilm. In a second part of the work, the study aimed at bringing some contribution to the understanding of the implication of the EPS in the adherence and cohesiveness properties of a Bacillus licheniformis biofilms, an agent of the paper industry surfaces colonization. The previously developed tools were useful to assess the level of contribution of each matrix component in the biofilm structural behavior in response to enzymatic actions. The combination of multiparametric analyses allowed to conclude about the possible interaction between functional amyloids and extracellular DNA as a strong complex that acts as an aggregative and adhesive factors to ensure biofilm stability and tridimensional structure

The Application of Genetic, In Silico and In Vitro Tools to Elucidate the Biology of a Functional Amyloid Fiber.

Curli are thin aggregative fimbriae produced by many Enterobacteriaceae as a structural component of biofilms. Curli share many biochemical and biophysical properties with amyloid fibers which are often associated with human neurological diseases including Alzheimer’s, mad cow, and Parkinson’s. However, curli are the product of a dedicated assembly system that consists of a complex gene regulatory network featuring CsgD; a secretion system including CsgG, CsgE, and CsgF; and the major and minor fiber subunits CsgA and CsgB. As a model system, many aspects of curli formation have been explored including subunit secretion, regulation, biological function, and amyloidogenesis. My work focused on the genetics of curli formation in Escherichia coli, the economic constraints on the evolution of CsgA and other extracellular proteins, and the in vitro amyloidogenesis of CsgA-His. I screened the Keio collection of single gene deletions to discover new genes that affect curli production. More than 300 genes modulate curli production including the sodium antiporter nhaA, a regulator of the glycine cleavage system gcvA, multiple LPS biosynthesis genes, and genes involved in many fundamental cellular processes. This analysis suggests that curli production is part of a highly regulated and complex developmental pathway. The regulation of glyA by CsgD and the curli phenotype of gcvA focused my attention on the amino acid composition of CsgA. CsgA is incredibly rich in glycine and serine. As simple amino acids, both are inexpensive to synthesize. Consequently, CsgA is relatively cheap to produce on a per unit basis. Strikingly, other extracellular proteins including those in Escherichia coli, Pseudomonas syringae, Mycobacterium tuberculosis, Saccharomyces cerevisiae, and other microbes are also inexpensive relative to intracellular proteins. Since extracellular proteins are often lost to the environment, evolution has in turn selected them for increased economy to counteract lost resources. Finally, we studied the in vitro amyloid formation of CsgA-His. Like disease-associated amyloids, CsgA-His bound Thioflavin T upon polymerizing into fibers, reacted with an amyloid specific antibody, self seeded, and displayed other aspects of amyloid formation. Collectively, this work sheds new light on the biology of the functional amyloid fiber curli and hopefully will beget novel directions of inquiry.Ph.D.Molecular, Cellular, and Developmental BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/78834/1/dnlsmith_1.pd

Comprehension of the mechanisms of action of antimicrobial molecules using nanobiotechnologies

Mon travail de thèse consiste à utiliser les techniques de Microscopie à Force Atomique (AFM) pour étudier les microorganisms pathogènes, et leurs interactions avec des antimicrobiens. Ces dernières décennies, la résistance microbienne a augmenté de façon dramatique. Les bactéries et levures pathogènes sont à l'origine d'infections mettant en jeu la vie de patients. Il y a donc deux urgences : la première est de trouver de nouveaux antimicrobiens; pour cela il faut acquérir des données fondamentales sur la paroi des microorganismes, afin d'identifier des cibles. La deuxième urgence est donc de développer des nouvelles techniques pour explorer la paroi des. Durant cette thèse nous avons donc utilisé l'AFM, adapté aux conditions biologiques. Un avantage de l'AFM est la possibilité de travailler en liquide, ce qui nous a permis d'imager l'élongation de cellules de P. aeruginosa traitées avec un antibiotique, ainsi que la disparition de la capsule de K. pneumoniae traitée avec de la colistine. L'AFM est aussi une machine de force qui enregistre des courbes de force permettant d'accéder aux propriétés nanomécaniques et d'adhésion des cellules. Nous avons ainsi observé les modifications des propriétés adhésives de la levure C. albicans traité avec de la caspofongine. Enfin il est possible de fonctionnaliser des pointes AFM avec des biomolécules ; cette stratégie nous a permis de localiser des protéines spécifiques à la surface de levures et cellules animales, et d'étudier la paroi de P. aeruginosa traité par un antibactérien innovant, le Cx1. Pour conclure, cette thèse a permis d'adresser spécifiquement la contribution de la biophysique en microbiologie clinique.My PhD work consists in using Atomic Force Microscopy (AFM) techniques to study pathogenic microorganisms, and to probe their interactions with antimicrobials. During the last three decades, microbial resistance has dramatically increased and spread around the world. Pathogenic bacteria and yeasts are the cause of life-threatening infections in some patients. There are therefore two emergencies; the first one is to find new antimicrobial molecules; for that, it is mandatory to get further knowledge on the microbial cell wall. Therefore the second emergency is to develop new techniques to explore microbial surfaces. During my PhD, we took advantage of a technology coming from physics, and adapted to biological conditions, AFM. An advantage of AFM is the possibility to work in liquid on living cells, which allowed us to image the elongation of cells of P. aeruginosa treated by ticarcillin, and the removal of capsular polysaccharides from K. pneumoniae upon treatment with colistin. AFM is also a force machine, able to record force distance curves that give access to nanomechanical and adhesive properties of cells. We could observe the modifications of the adhesive properties of the yeast C. albicans, treated by caspofungin. Finally it is possible to functionalize AFM tips with biomolecules; we used this strategy to localize specific proteins at the surface of living yeasts and mammalian cells, and to study the cell wall of P. aeruginosa treated by an innovative antibacterial, Cx1. In conclusion, during my PhD, we especially addressed the contribution of biophysics in clinical microbiology

Student Research Colloquium Proceedings 2008

2008 Student Research Colloquium proceedings include the following: a schedule of the day\u27s events, acknowledgement of research sponsors, the day\u27s program, conference presentation abstracts, student presenter index, research sponsor index, planning committee, poster and paper presentation judges, registration desk, sponsors, and donors, map of Atwood Memorial Center. Invited Alumni Keynote Address: Research in Action: Helping People included the following SCSU alumni panelists: Keesha Gaskins, Executive Director of Minnesota Women\u27s Political Caucus; Bob Goff, Co-founder and owner of Goff & Howard, Inc., a public relations firm; Amy Schultz, Geropsychologist from OPAL Institute, Oregon Passionate Aging and Living. These distinguished alumni will discuss how research helps them do their jobs. Panelists will describe their careers and answer questions about the secret to their success

Synthesis of new pyrazolium based tunable aryl alkyl ionic liquids and their use in removal of methylene blue from aqueous solution

In this study, two new pyrazolium based tunable aryl alkyl ionic liquids, 2-ethyl-1-(4-methylphenyl)-3,5- dimethylpyrazolium tetrafluoroborate (3a) and 1-(4-methylphenyl)-2-pentyl-3,5-dimethylpyrazolium tetrafluoroborate (3b), were synthesized via three-step reaction and characterized. The removal of methylene blue (MB) from aqueous solution has been investigated using the synthesized salts as an extractant and methylene chloride as a solvent. The obtained results show that MB was extracted from aqueous solution with high extraction efficiency up to 87 % at room temperature at the natural pH of MB solution. The influence of the alkyl chain length on the properties of the salts and their extraction efficiency of MB was investigated