Chapter: 1 Introduction Genus Salmonella is a Gram-negative rod shaped facultative anaerobic bacteria that can survive inside the host macrophages and cause persistent infection. Salmonella Typhimurium, Salmonella Typhi and Salmonella Enteritidis are the serovars, which belong to the Salmonella enterica species. S. Typhi causes typhoid fever in humans. S. Typhimurium is one of the important causes for food poisoning in humans. It causes typhoid like fever in mice and serves as a good model system to study Salmonella pathogenesis. Salmonella infection occurs via the orofecal route following which it invades the intestinal mucosa through several ways, namely by antigen sampling M cells, CD18+ macrophages present in the intestinal lumen or via a forced entry in the non phagocytic enterocytes. Upon entry Salmonella resides in an intracellular phagosomal compartment called the Salmonella containing vacuole (SCV). The SCV only transiently acquires endocytic markers like TfnR, EEA1, Rab4, Rab5, Rab11 and Rab7. It eventually uncouples from the endocytic pathway to avoid lysosomal fusion and ultimately reaches the golgi apparatus achieving a perinuclear position. The mechanisms by which phagocytes kill the virulent Salmonella are not completely understood, however the role of nicotinamide-adenine dinucleotide phosphate (NADPH) phagocytic oxidase system has been strongly implicated. The generation of reactive oxygen species (ROS) occurs via a membrane-bound flavocytochrome b558, consisting of two phagocytic oxidase components (gp91phox and p22phox) and four cytosolic components, p40phox, p47phox, p67phox, and a GTP-binding Rac protein. Further, professional phagocytes like macrophages generate nitric oxide (NO) that acts as a potent agent to limit the growth of many intracellular pathogens including Salmonella. Chapter:2 Resistance to host Nitrosative stress in Salmonella by quenching L-arginine. Arginine is a common substrate for both inducible nitric oxide synthase (iNOS) and arginase. The competition between iNOS and arginase for arginine contributes to the outcome of several parasitic and bacterial infections. Salmonella infection in macrophage cell line RAW264.7 induces iNOS. Because the availability of L-arginine is a major determinant for nitric oxide (NO) synthesis, we hypothesize that in the Salmonella infected macrophages NO production may be regulated by arginase. Here we report for the first time that Salmonella up-regulates arginase II but not arginase I isoform in RAW264.7 macrophages. Blocking arginase increases the substrate L-arginine availability to iNOS for production of more nitric oxide and perhaps peroxynitrite molecules in the infected cells allowing better killing of virulent Salmonella in a NO dependent manner. RAW264.7 macrophages treated with iNOS inhibitor aminoguanidine reverts the attenuation in arginase blocked condition. Further, the NO block created by Salmonella was removed by increasing concentration of L-arginine. In the whole-mice system arginase I, although constitutive, is much more abundant than the inducible arginase II isoform. Inhibition of arginase activity in mice during the course of Salmonella infection reduces the bacterial burden and delays the disease outcome in a NO dependent manner. Chapter:3 Hrg (hydrogen peroxide resistant gene), a LysR type transcriptional regulator confers resistance to oxidative stress in Salmonella LysR type transcriptional regulators are one of the key players that help bacteria adapt to different environments. We have christened STM0952, a putative LysR type transcriptional regulator in Salmonella enterica serovar Typhimurium as the hydrogen peroxide resistance gene (hrg). By generating a knock out of the hrg gene, we demonstrate that the hrg mutant serovar Typhimurium is sensitive to oxidative products of the respiratory burst, specifically to hydrogen peroxide. The hrg mutant is profoundly attenuated in the murine model of infection and shows decreased intracellular proliferation in macrophages. It was also found to induce increased amount of reactive oxygen species and co-localization with gp91phox in the macrophage cell line, when compared to the wild type. An overproducing strain of this gene showed a survival advantage over the wild type Salmonella under hydrogen peroxide induced stress condition. Microarray analysis suggested the presence of a Hrg regulon, which is required for resistance to the toxic oxidative products of the reticulo-endothelial system. Chapter:4 Importance of the host oxidative stress in antigen presentation and its modulation by Salmonella: Role of TLR Synthetic CpG containing oligodeoxynucleotide TLR-9 agonist (CpG ODN) activates innate immunity and can stimulate antigen presentation against numerous intracellular pathogens. We report that Salmonella Typhimurium growth can be inhibited by the CpG ODN treatment in the murine dendritic cells. This inhibitory effect was shown to be mediated by an increased reactive oxygen species (ROS) production. We further show that the CpG ODN treatment of the dendritic cells during Salmonella infection leads to a ROS dependent increased antigen presentation. In addition, TLR-9 signaling inhibitor was able to inhibit the CpG ODN mediated increased antigen presentation, ROS production and pathogen killing. These data indicate that CpG ODN can improve the ability of the murine dendritic cells to contain the growth of the virulent Salmonella through ROS dependent killing and could as well be used as an effective adjuvant in vaccines against Salmonella infection.

The primary aim of the work presented in this thesis was to change the inducer specificity of the DntR protein in order to improve the response to DNT. The long-term goal is to use this protein in a biosensor for DNT, a signature compound for detection of the explosive TNT. Another aspect of this work was to understand the mechanisms of inducer binding and how the binding of an inducer molecule changes the DntR structure into a state that triggers transcriptional activation. In the papers included in this thesis the inducer specificity of wt DntR has been investigated under different conditions. The functional effects of specific mutations have also been investigated, in some cases in combination with structure determination using X-ray crystallography. In addition, structural data offering insights into the details of inducer binding and conformational changes upon inducer binding are presented and discussed in terms of mechanisms for transcriptional activation by DntR. Furthermore, a directed evolution strategy was employed in order to find variants of DntR with improved response to DNT. A variant with a large improvement in the DNT response was isolated and characterized. In optimized growth conditions, this DntR variant had a nearly 10-fold increase in fluorescence in response to DNT compared to wt DntR. Specific substitutions found in this DntR variant are suggested to be important for changing the inducer response. / Syftet med denna avhandling har varit att förbättra förmågan hos proteinet DntR att upptäcka DNT. Det långsiktiga målet har varit att använda DntR i en biosensor för att upptäcka sprängämnet TNT, som avger DNT som en ”signaturmolekyl”. En annan aspekt har varit att bättre förstå den detaljerade mekanismen för hur DntR fungerar. DntR är ett protein som binder till en viss DNA sekvens (promotor) och reglerar hur gener intill denna promotorsekvens läses av. När en inducerande molekyl som t.ex. DNT binder till DntR förändras proteinets struktur på ett sådant sätt att DntR kan aktivera transkription av de gener som finns intill promotor-sekvensen. För att mäta hur DntR reagerar på olika inducerande molekyler har DntR uttryckts i bakterien Escherichia coli, som också innehållit promotorn som DntR binder till. Intill promotorn sitter en gen som kodar för proteinet GFP. När en inducerande molekyl binder till DntR, slås avläses gfp-genen, och det fluorescerande proteinet GFP produceras. Ju mer GFP som produceras i cellerna, desto högre fluorescens kan uppmätas när cellerna analyseras.   I de artiklar som presenteras i avhandlingen har vi undersökt hur olika substitutioner i DntR proteinet påverkar specificiten och sensitiviteten och hur dessa egenskaper kan påverkas av olika experimentella faktorer. Effekten av substitutioner har relaterats till strukturdata, där bilder av hur proteinet ser ut på molekylär nivå har tagits fram. Dessutom presenteras även en bild av hur DntR förändras beroende på om inducerande molekyler är bundna eller inte. En sådan strukturbild ökar förståelsen för de mekanismer som gör att bindning av en inducerande molekyl orsakar en förändring av formen hos DntR på så sätt att avläsning av gener kan aktiveras. Vi har också använt en metod där evolutionära processer härmats för att få fram varianter av DntR med förbättrad respons till DNT. En variant med en drastisk ökning av DNT-responsen har isolerats, och dess egenskaper har karaktäriserats. / At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript

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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