Yes / Burkholderia spp. rely on N-acyl homoserine lactone as quorum-sensing signal molecules which coordinate their phenotype at the population level. In this work, we present the whole genome of Burkholderia sp. strain A9, which enables the discovery of its N-acyl homoserine lactone synthase gene. / UM High Impact Research Grants (UM-MOHE HIR grant UM C/625/1/HIR/MOHE/CHAN/01, H-50001-A000001 and UMMOHE HIR Grant UM C/625/1/HIR/MOHE/CHAN/14/1, H-50001- A000027)

This thesis entitled ‘ Unusual Acylation Properties of Type II Fatty Acid Biosynthesis Acyl Carrier Proteins’ describes the discovery of self-acylation and malonyl transferase activity in acyl carrier proteins involved in type II fatty acid biosynthesis and assigns a physiological role to these processes inside the cellular milieu. Acyl carrier protein (ACP) is one of the most abundant proteins present inside the cell and almost 4% enzymes require it as a cofactor. Acyl carrier proteins can exist either as discrete proteins or as domains of large functional proteins. They function in a variety of synthases as central molecules to which growing acyl intermediates and nascent product molecules are covalently tethered during the elongation and modification steps required to produce the final product. A prototypical bacterial ACP is composed of 70-80 amino acids and is generally expressed in the apo form. It is post-translationally modified to active holo form by the addition of 4'-phosphopantetheine moiety to an absolutely conserved serine residue in a reaction catalyzed by holo-ACP synthase or 4'-phosphopantetheine transferase. Chapter 1 surveys literature related to carrier proteins inside the cell and describes the thesis objective. It also presents an overview of the acyl carrier proteins and their involvement in various metabolic pathways inside the cell. The chapter details the structural organization of acyl carrier proteins from various sources revealing the conservation in their structure and also details the molecular basis of interaction of ACP with other enzymes inside the cell. The discovery of unusual self-acylation property in acyl carrier proteins involved in polyketide biosynthesis and its absence in acyl carrier proteins involved in fatty acid biosynthesis prompted me to investigate the reasons for this selective behavior. Discovery of self-acylation property in acyl carrier proteins Plasmodium falciparum and chloroplast targeted Brassica napus acyl carrier proteins involved in type II fatty acid biosynthesis and the mechanism of this reaction forms the basis of Chapter 2. In this chapter it has been shown that self-acylation property is intrinsic to a given acyl carrier protein and is not dependent on the pathway in which it is involved. Based on primary sequence analysis and site directed mutagenesis studies presence of an aspartate/glutamate has been identified to be critical for the self-acylation event. Furthermore, it has also been shown that the self-acylation event in type II fatty acid biosynthesis acyl carrier proteins is highly specific in nature employing only dicarboxylic acid –CoAs as substrates unlike the polyketide biosynthesis acyl carrier proteins which utilize both dicarboxylic acid and β-keto acid thiol ester -CoAs as substrates. The detailed kinetics of these reactions has also been worked out. Combining all the results a plausible mechanism for the self-acylation reaction has been proposed. Chapter 3 describes the discovery of a novel malonyl transferase behavior in acyl carrier proteins involved in type II fatty acid biosynthesis. Malonyl transferase property in ACPs of type II FAS from a bacterium (Escherichia coli), a plant (Brassica napus) and a parasitic protozoon (Plasmodium falciparum) were investigated to present a unifying paradigm for the mechanism of malonyl transferase behavior in ACPs. Identification of malonyl transferase property in Plasmodium falciparum ACP and Escherichia coli ACP (EcACP) and the absence of this property in Brassica napus ACP has been described in this chapter. Detailed investigations demonstrated that presence of an arginine or a lysine in loop II and an arginine or glutamine at the start of helix III as the residues that are critical for the transferase activity. In order to assign a physiologic function to these unusual acylation properties, fabD(Ts) mutant strain of Escherichia coli was utilized for heterologous complementation by the various wild type and mutant ACPs that are able to catalyze either or both of the activities. Growth of the mutant strain at non-permissive temperature, when complemented with ACPs catalyzing both the reactions confirmed that these properties have a physiologic relevance. Extensive mutagenesis experiments in conjunction with complementation studies allowed me to propose a plausible mechanism on how the self-malonylation and malonyl transferase properties operate in tandem. Chapter 4 describes the thermodynamic characterization of self-acylation process using Isothermal Titration Calorimetry. Isothermal Titration Calorimetric studies on the binding of malonyl, succinyl, butyryl and methylmalonyl –CoA to Plasmodium falciparum and Brassica napus acyl carrier proteins were performed to investigate the role of thermodynamic parameters in the specificity of self-acylation reaction. Calculation of the parameters showed that the thermodynamics does not control the self-acylation reaction. The evolution of self-acylation property in various acyl carrier proteins and its possible significance in the evolution of various metabolic events is described in Chapter 5. Extensive bioinformatics search was performed and phylogenetic analysis on acyl carrier proteins from 60 different taxa was done using the MEGA4 program. Analysis showed that this property was first found in cyanobacterium. Later, during the course of evolution this property was lost in most acyl carrier proteins, and was retained either in acyl carrier proteins that are targeted to organelles of cyanobaterial orgin viz. apicoplast in apicomplexans and chlorplasts in plants or in acyl carrier proteins involved in secondary metabolic events such as polyketide biosynthesis. Chapter 6 summarizes the findings of the thesis. Acyl carrier protein from Plasmodium falciparum, Brassica napus and Escherichia coli were characterized for their self-acylation and malonyl transferase properties and a combined mechanism for these two properties is proposed. The work done also provides an in vivo rationale to these in vitro processes. Furthermore, the evolutionary significance of the self-acylation behavior is also discussed in the thesis. The thesis also probes into the thermodynamics of the self-acylation reaction in Plasmodium falciparum and Brassica napus acyl carrier proteins. Thus, the thesis adds a new dimension to the much unexplored ACP biology and paves the way to study in vivo roles of these processes in detail. Appendix I describes the Isothermal Titration calorimetric characterization of binding of various acyl-PO4 molecules to Escherichia coli PlsX (Acyl-phosphate acyltransferase). PlsX, the first enzyme of phosphatidic acid biosynthesis pathway catalyzes the conversion of acyl-ACP into acyl-PO4, which is further used by other enzymes leading to the formation of phosphatidic acid. ITC results presented in this section show that longer chain length acyl-PO4 molecules show better binding to PlsX, as compared to the smaller ones demonstrating that long chain acyl molecules serve as better substrates for phosphatidic acid synthesis.

Le « quorum sensing » (QS) est un moyen de communication bactérienne impliquant des petites molécules appelées auto-inducteurs qui au-delà d’un certain seuil de concentration induisent une synchronisation de l’expression génétique au sein de la communauté bactérienne. Ce mécanisme est impliqué dans plusieurs processus bactériens tels que la luminescence, la formation du biofilm, ce qui en fait une cible privilégiée pour l’inhibition du biofilm bactérien nuisible aux activités humaines. Plusieurs systèmes QS ont été identifiés ; les plus étudiés sont le système AHL (acyl homoserine lactone) et le système AI2 (auto inducteur 2). L’objectif principal de cette thèse est de caractériser le(s) système(s) QS de Shewanella woodyi, une bactérie marine luminescente capable de coloniser rapidement une surface et de former un biofilm. L’utilisation de biosenseurs de référence et des expériences de LC-MS ont montré que S. woodyi synthétise la C8-HSL et l’AI2. La mutation des gènes impliqués dans la synthèse ou la détection des HSL abolit la luminescence mais n’affecte pas la formation du biofilm. De plus, le système AI2 ne semble pas impliqué dans la luminescence et la formation de biofilm de S. woodyi. L’absence d’un récepteur d’AI2 suggère que cette molécule n’a pas un rôle régulateur et qu’elle ne serait qu’un produit secondaire du métabolisme cellulaire. Ce travail a donc permis de caractériser les 2 principaux systèmes QS de S. woodyi et pourrait permettre d’en faire un nouveau biosenseur marin. / Quorum sensing (QS) is a bacterial communication system involving small molecules called autoinducers which above a threshold concentration, induce the synchronization of genes expression within the bacterial community. This mechanism is involved in several bacterial processes such as luminescence and biofilm formation, making it a preferred target for the inhibition of bacterial biofilm harmful to human activities. Several QS systems have been identified; the most studied ones are the AHL system (acylhomoserine lactone) and the AI2 system (autoinducer 2). The main objective of this thesis is to characterize the QS system (s) of Shewanella woodyi, a luminescent marine bacterium able to rapidly colonize a surface and form a biofilm. The use of reference biosensors and LC-MS experiments have shown that S. woodyi synthesizes C8-HSL and AI2. The mutation of the genes involved in the synthesis or detection of HSL abolishes luminescence but does not affect the biofilm formation. Moreover, the AI2 system does not appear to be involved in the luminescence and biofilm formation of S. woodyi. The absence of an AI2 receptor suggests that this molecule does not have a regulatory role and that it is only a secondary product of cellular metabolism. This work has allowed the characterization of the 2 main QS systems of S. woodyi, which could make this strain a new marine biosensor.

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Depuis quelques décennies, les bactéries pathogènes multi-résistantes aux antibiotiques sont de plus en plus répandues dans le monde. Cette situation a suscité le besoin et l'intérêt de trouver des médicaments antibactériens avec de nouvelles cibles potentiels. La découverte des systèmes de communication de type quorum sensing (QS) régulant la virulence bactérienne représente une des cibles privilégiées pour contrôler les bactéries pathogènes autrement qu’en interférant avec leur croissance bactérienne. Dans l’écosystème naturel, un grand nombre d'organismes (Eucaryotes et Procaryotes) co-existent en synthétisant chacun de leur côté des métabolites secondaires. Les plantes, étant en permanence en contact avec des bactéries, synthétisent des métabolites secondaires capables d’inhiber l’expression des gènes de virulence chez les bactéries sans pour autant affecter ni leur croissance ni leur viabilité. Notre objectif a été de contribuer à la valorisation de la biodiversité malgache en identifiant des plantes et en y isolant les composés actifs présentant une capacité à perturber le mécanisme de QS chez P. aeruginosa PAO1, une bactérie pathogène opportuniste de l’homme, des animaux et des plantes. Dans ce but, nous avons tout d’abord réalisé un criblage d’activité anti-QS de différents flavonoïdes commerciaux. De ce criblage, la narigenine et la naringine ont été sélectionnées pour être les molécules de contrôle positif et négatif des tests d’activité anti-QS, respectivement. Par la suite, 4 espèces de Dalbergia endémique de Madagascar ont fait l’objet de criblage pour leur activité anti-QS. Ce travail a fait ressortir l’activité anti-QS très intéressante de l’écorce de D. trichocarpa à partir de laquelle nous avons isolée le composé actif nommé la coumarate de l’aldéhyde-oléanolique (OALC). Le contrôle naringénine et l’OALC ne présente aucun effets inhibiteurs sur la croissance bactérienne de P. aeruginosa PAO1 et sur l’expression du gène QS-indépendant aceA suggérant une activité d’inhibition spécifiquement liée au QS. Cependant, ces deux molécules présentent des spectres d’inhibition différente. En effet, les deux molécules diffèrent dans le sens que la naringenine n’inhibe pas l’expression du gène gacA et la motilité de type twitching contrairement à l’OALC. Ces résultats suggèrent que l’OALC et la naringénine représente des candidats potentiels pour des investigations in vivo quant à leur effet anti-QS et anti-biofilm sur des modèles infectieux d’organismes supérieurs. Par ailleurs, ils démontrent la richesse des plantes malgaches comme sources de nouvelles molécules anti-virulence ainsi que l’importance de telle investigation afin de renforcer notre arsenal thérapeutique en composé antibactérienne dans la lutte continuelle contre les bactéries pathogènes/Since few decades, multidrug resistant bacteria spread all over the world. This situation gives rise to the need and interest in finding antibacterial drugs with novel potent target. Discovery of communication system termed Quorum Sensing (QS) which regulate bacterial virulence factor represent privileged target in another way than interfering with bacterial growth. In natural ecosystem, many organisms (Eukaryotes and Prokaryotes) produce secondary metabolites. As plants are permanently in contact with bacteria, they have synthetized secondary metabolites which inhibit bacterial virulence gene expression without affecting bacterial viability. Our goal was to contribute to the valorization of Malagasy biodiversity and specifically to identify plants and isolate bioactive compound presenting ability to disrupt QS mechanism in P. aeruginosa, opportunistic pathogen bacteria in plants, animals and human. In this purpose, screening of commercial available flavonoids has been firstly carried out. From this screening, naringenin and naringin have been selected to be used as positive and negative QS inhibitor controls, respectively. Subsequently, Four Malagasy endemic Dalbergia species have been screened for their anti-QS activity. This work pointed out the interesting anti-QS activity of D. trichocarpa bark extract which led to the isolation of oleanolic aldehyde coumarate (OALC) as one major bioactive compound. At the concentration tested, naringenin and OALC did not affect P. aeruginosa PAO1 viability and didn’t reduce QS-independent aceA gene expression suggesting a specific anti-QS activity. However, these two compounds present different inhibition spectrum. Indeed, naringenin didn’t inhibit gacA gene expression and twitching motility contrarily to OALC. These results suggest that OALC and naringenin represent potent candidates for in vivo investigations in their anti-QS and anti-biofilm activity onto eukaryotes infectious model. Besides, this finding demonstrated the potent source for novel anti-virulence compounds of Malagasy flora and the importance of this kind of research to strengthen our antimicrobial therapeutic arsenal with the ongoing struggle against bacterial infection. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Plants are continuously confronted with a multitude attack either abiotic but also biotic in nature. Interestingly, despite the abundance of bacteria that plant has to face, only few are able to induce death or disease in the host plant. It is therefore likely that, in addition to secondary metabolites with antimicrobial properties, plants also synthesize secondary metabolites which are able to inhibit the expression of virulence genes in bacteria without affecting either growth or viability, which allows plants to host willingly or not bacterial populations. This work focuses on the identification of such metabolites in Malagasy plants (genera Dalbergia and Combretum) and the demonstration of their inhibitory effect on the expression of virulence genes in two different pathosystems: Rhodococcus fascians (a phytopathogen) and Pseudomonas aeruginosa (an opportunistic pathogen). Thus, two metabolites were isolated using a combination of chromatographic techniques coupled with tests that evaluate the expression of certain genes involved in the virulence mechanisms of these bacteria. The first is a new prenylated isoflavanone, named perbergin, isolated from the bark extract of D. pervillei. It was shown that the perbergin target attR gene expression, encoding a LysR-type transcriptional regulator that plays a key role in regulating the expression of virulence genes of R. fascians and the transition from an epiphytic to a pathogenic lifestyle. Therefore, we have also shown that the expression of all virulence genes known to date in R. fascians is also affected while the expression of genes involved in epiphytic fitness of the bacteria is not altered. In addition, the application of perbergin at the time of infection of plants susceptible to R. fascians shows that this molecule reduces in vivo the virulence of R. fascians, highlighting the potential of perbergin as an anti-infective agent. The second is a flavonoid known as catechin, isolated from the bark extract of C. albiflorum. Catechin significantly inhibits the expression of genes that regulate the mechanism of quorum sensing in P. aeruginosa such as lasI, LasR, rhlI and rhlR but also lasB and rhlA which expression depends on quorum sensing. Therefore, the production of virulence factors such as pyocyanin and elastase is significantly affected. Because of the limited number of our arsenal of antibiotics and their increasing ineffectiveness, the identification of these compounds create a path to an alternative in the fight against pathogenic bacteria and multidrug resistance of pathogenic bacteria to antibiotics. Our results also demonstrate the richness of Malagasy plants as (re)sources of new therapeutic molecules and the importance of widening the range of bacterial targets to be investigated to develop new strategies to fight within the endless war that we are waging against bacteria pathogens.<p><p>Les plantes sont continuellement confrontées à une multitude d’attaques qu’elles soient de nature abiotique ou surtout biotique. Il est intéressant de noter que malgré la multitude de bactéries auxquelles les plantes doivent faire face, seules quelques unes sont capables d’induire la mort ou une maladie chez la plante hôte. Il est dès lors fort probable que, outre les métabolites secondaires ayant des propriétés antimicrobiennes, les plantes synthétisent également des métabolites secondaires capables d’inhiber l’expression des gènes de virulence chez les bactéries sans toutefois affecter ni leur croissance ni leur viabilité, ce qui permet aux plantes de contenir les populations bactériennes qu’elles hébergent de gré ou de force. Ce travail porte sur l’identification de ce type de métabolites dans des plantes malgaches (genres Dalbergia et Combretum) et la démonstration de leurs effets inhibiteurs sur l’expression de gènes de virulence chez deux pathosystèmes différents: Rhodococcus fascians (un phytopathogène) et Pseudomonas aeruginosa (un pathogène opportuniste). Ainsi, deux métabolites ont été isolés en utilisant une combinaison de techniques chromatographiques couplées avec des tests qui évaluent l’expression de certains gènes impliqués dans les mécanismes de virulence de ces bactéries. Le premier est un nouvel isoflavanone prénylé, nommé perbergine, isolé à partir de l’extrait d’écorces de D. pervillei. Il a été montré que la perbergine cible l’expression du gène attR, codant un régulateur transcriptionnel de type LysR qui joue un rôle clé dans la régulation de l’expression des gènes de virulence de R. fascians et qui assure la transition entre un mode de vie épiphyte et le mode pathogène. En conséquence, nous avons également montré que l’expression de l’ensemble des gènes de virulence connu à ce jour chez R. fascians est également affectée alors que l’expression de gènes impliqués dans l’aptitude épiphyte de la bactérie n’est pas altérée. Par ailleurs, l’application de perbergine au moment de l’infection de plantes sensibles à R. fascians montre que cette molécule atténue la virulence de R. fascians in vivo, mettant en exergue le potentiel de la perbergine comme agent anti-infectieux. Le deuxième est un flavonoïde, connu sous le nom de catéchine, isolé de l’extrait d’écorces de C. albiflorum. La catéchine inhibe significativement l’expression des gènes régulateurs du mécanisme du quorum sensing chez P. aeruginosa tels que lasI, lasR, rhlI et rhlR et également lasB et rhlA dont l’expression dépend du quorum sensing. En conséquence, la production des facteurs de virulence tels que la pyocyanine et l’élastase est significativement affectée. Compte tenu de l’appauvrissement de notre arsenal d’antibiotiques et de leur inefficacité croissante, l’identification de ces composés ouvre une voie alternative de lutte contre les bactéries pathogènes et la multirésistance des bactéries pathogènes aux antibiotiques. Nos résultats démontrent également la richesse des plantes malgaches comme (res)sources de nouvelles molécules thérapeutiques et l’importance d’élargir le champ des cibles bactériennes à investiguer pour développer de nouvelles stratégies de lutte dans la guerre sans fin que nous menons contre les bactéries pathogènes. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished