Wang Xiaofei. / "June 2002." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (p. 152-167). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Le rôle des acides biliaires a évolué ces dernières années passant de simples molécules solubilisatrices des lipides à des composés à activité métabolique. En plus de leur fonction dans l’absorption des lipides post-repas, ils ont été montrés comme stimulant de nombreuses voies de signalisation modulant l’expression de gènes clefs du métabolisme et de nombreux mécanismes physiologiques via l’activation de récepteurs spécifiques tels que les récepteurs « Farnesoid X receptor » (FXR) et le récepteur membranaire couplé à une protéine G, TGR5. La protéine TGR5 codée par le gène GPBAR1, aussi connue sous le nom de « G-protein-membrane-type receptor for bile acids » (M-BAR) est le premier récepteur couplé à une protéine G spécifique aux acides biliaires ayant été mis en évidence. Cette protéine est exprimée dans de nombreux tissus clefs du métabolisme énergétique tels que les cellules L intestinales, le tissu adipeux, les reins, le muscle squelettique et le pancréas. En réponse à la fixation des acides biliaires au récepteur TGR5, celui-ci va être internalisé et sa sous-unité GαS va être libérée. Ce mécanisme va ensuite activer l’adénylate cyclase et augmenter la production d’AMPc à l’origine de l’activation des voies de signalisations liées à la protéine kinase A (PKA). Une fois activée, la PKA va induire la phosphorylation des protéines « cAMP-response element-binding » (CREB) et permettre la modulation de l’expression de gènes cibles.Ces dernières années de nombreux travaux ont eu pour but d’étudier le rôle du récepteur TGR5 dans le métabolisme. Chez la souris, l’activation du récepteur TGR5 stimule la dépense énergétique dans le tissu adipeux brun et dans le muscle squelettique et prévient le développement de l’obésité et de l’insulino-résistance induites par un régime riche en graisses. Le récepteur TGR5 est également impliqué au niveau des cellules L intestinales sécrétrices du GLP-1. Il y joue un rôle essentiel dans l’homéostasie glucidique via la régulation de l’activité pancréatique, des sécrétions de l’insuline et du glucagon, de l’inhibition de la vidange gastrique ou encore de la modulation des messages de satiété via des voies neuroendocrines. TGR5 présente également des fonctions immunologiques avec une expression connue dans les cellules de l’immunité telles que les monocytes, les macrophages alvéolaires ou encore les cellules de Kupffer. TGR5 a également été mis en évidence comme régulateur des mécanismes d’inflammations via les macrophages avec une diminution de l’expression des cytokines pro-inflammatoires. A l’opposé, l’activation de TGR5 serait impliquée dans de nombreux processus pathologiques tels que, le développement de carcinomes gastro-intestinaux, les pancréatites, la lithiase biliaire, suggérant un rôle potentiel du récepteur TGR5 dans la régulation de voies de signalisation responsables de la prolifération et de la mort cellulaire [...] / Bile acids (BAs) have evolved over the years from being considered as simple lipid solubilizers to metabolically active molecules. In addition to their function in dietary lipid absorption, they have also been shown to activate farnesoid X receptor (FXR) and TGR5 receptors to initiate signaling pathways and regulate metabolic gene transcription. TGR5 (encoded by the GPBAR1 gene), also known as G-protein-membrane-type receptor for bile acids (M-BAR) or G-protein-coupled bile acid receptor 1 (GPBAR1), was the first identified G-protein coupled receptor specific for bile acids. In normal individuals, the highest level of GPBAR1 mRNA expression was reported in the gallbladder, placenta and spleen, followed by moderate expression in other tissues including lungs, liver, stomach, small intestine and adipose tissue, with a relatively low level of expression in kidney, skeletal muscles and pancreas. In response to binding of BAs to the ligand-binding pocket of the TGR5 protein, the TGR5 receptor is internalized and the GαS subunit is released. This mechanism leads to activation of adenylate cyclase and an increase in cAMP production resulting in induction of the protein kinase A (PKA) pathway. Subsequently, PKA phosphorylates the cAMP-response element-binding protein (CREB) and enhances the transcription of its target genes in response to extracellular signals.To date, extensive work has been done to investigate the role of TGR5 in metabolism. In rodents, BA-activated TGR5 receptor stimulates energy expenditure in brown adipose tissue and skeletal muscle and prevents obesity and insulin resistance induced by a high fat diet. TGR5 is also implicated in intestinal L-cells secreted GLP-1, which plays an essential role in glucose homeostasis through the stimulation of glucose-dependent-insulin-secretion and inhibition of glucagon secretion, inhibition of gastric emptying and increasing satiety through neuroendocrine pathways. In terms of the immunological function of TGR5, it is now known that TGR5 is expressed in several immune cells such as monocytes, alveolar macrophages and Kupffer cells. The beneficial effects of TGR5 on macrophage-driven inflammation include reduced proinflammatory cytokine expression, thus protecting against atherosclerosis and liver steatosis. On the contrary, TGR5 activation has also been implicated in itch and analgesia, gastrointestinal-tract cell carcinogenesis, pancreatitis, and cholelithiasis, suggesting a potential role for TGR5 as a regulator of signal transduction pathways responsible for cell proliferation and apoptosis. BAs may also influence islet function via both direct and indirect mechanisms as recent studies have shown that Farnesoid X receptor (FXR) is expressed by pancreatic beta cells, and regulates insulin signaling in cultured cell lines. Kumar et al., [14] also reported that the TGR5 agonists INT-777 + oleanolic acid (OA) stimulated glucose-mediated insulin release via TGR5 activation, also in cultured cells. Still, little is known about the regulation of TGR5 expression or its involvement in pancreatic hormone secretion in response to physiological or pathological conditions such as T2D, as these studies have been performed mainly in cultured cell lines. In these contexts, the biological function of TGR5 remains enigmatic. The aim of the present study was first to establish the specific expression of TGR5 in human pancreatic islet cell subtypes. Then, a cross-sectional cohort of human islets isolated from individuals with various degrees of insulin resistance was exploited to determine if TGR5 expression and function was modified in T2D. Finally to determine if targeting TGR5 is clinically relevant, human islets were treated in-vitro with a specific agonist of TGR5 or with siRNA directed against TGR5 and hormone secretion assessed to establish whether TGR5 activation or inhibition modulate pancreatic hormone secretion.

Pseudomonas aeruginosa is an opportunistic human pathogen that is now the leading cause of morbidity and mortality in immunocompromised individuals. Those suffering with the genetic disease cystic fibrosis (CF) commonly encounter P. aeruginosa infections. P. aeruginosa infection can present itself as an acute infection, which is characterised by highly virulent, "free-swimming" bacteria, or as a chronic infection associated with the formation of surface-adhered bacterial communities known as biofilms. The labyrinth of interconnecting signalling networks has meant that the regulatory mechanisms behind biofilm formation and virulence are largely undefined. In this dissertation, a single nucleotide polymorphism was identified within the gene, fusA1, encoding elongation factor G (EF-G). The mutation introduced minor structural changes to the protein which were likely to have functional repercussions in its involvement in protein synthesis. Phenotypic analysis revealed that the mutation conferred changes in both resistance and sensitivity to various antibiotics, as well as changes in motility, exoenzyme production, quorum sensing, metabolism, synthesis of biofilm-associated proteins and exopolysaccharide production. Most notably was the up-regulation of a major virulence determinant, the type three secretion system, typically characteristic of cells comprising an acute infection. Proteomic and transcriptomic profiling of the mutant strain provided an insight into the genetic basis behind these phenotypes, identifying the up-regulation of multidrug efflux systems and modulations to the chemotactic systems. This study also found links between several biological processes that were modulated in the mutant strain, such as crosstalk between sulfur metabolism, iron uptake and the oxidative stress response. In summary, the work presented in this dissertation highlights the susceptibility of fusA1 to spontaneous mutation and identifies a novel role for EF-G in bacterial virulence and antibiotic sensitivity, both of which have worrying implications for infection within the CF lung.

Xu Li. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 204-242). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Pseudomonas aeruginosa is a gram-negative pathogen that causes opportunistic infections in immunocompromised individuals. Whereas clinical isolates from acute infections are characterized by host cell cytotoxicity and motility, isolates from chronic infections are characterized by biofilm formation and persistence. The type III secretion system (T3SS) causes cytotoxicity by injecting effectors into host cells. T3SS gene expression is activated by ExsA, an AraC family transcriptional regulator. Transcription of exsA is controlled by two promoters, PexsC and PexsA, which are regulated by ExsA and the cAMP-Vfr system, respectively. Additional global regulatory systems also influence T3SS including the second messenger signaling molecule c-di-GMP and the RsmAYZ regulatory system. c-di-GMP signaling increases biofilm production and decreases acute virulence factor expression. A previous study found that c-di-GMP alters cAMP levels and affect cAMP-Vfr signaling. Other studies found that c-di-GMP signaling alters expression of the small non-coding regulatory RNAs, rsmY and rsmZ. The RsmAYZ post-transcriptional regulatory system regulates ExsA translation. We hypothesize that c-di-GMP regulates T3SS expression by altering exsA transcription through the cAMP-Vfr dependent PexsA promoter. Overexpression of YfiN, a c-di-GMP synthase, decreases T3SS reporter activity in PA103 and requires a functional GGDEF active site for full inhibition. Inhibition by YfiN does not require rsmYZ. YfiN expression decreases cAMP-Vfr signaling and coordinately inhibits PexsA-lacZ reporter activity. Consistent with the proposed model, YfiN expression in a vfr mutant does not further decrease T3SS reporter activity. These data indicate that the YfiN alters T3SS expression through transcriptional control of the cAMP-Vfr dependent PexsA promoter.

The gram-negative marine bacterium Vibrio parahaemolyticus is a pathogen and a common worldwide cause of seafood-associated gastroenteritis. When grown on a surface, V. parahaemolyticus undergoes a dramatic differentiation to an elongated, highly flagellated swarmer cell from the short rod typical of swimming cells. Swarming motility is a complex form of adaptation to growth on a surface, and we developed a set of microarray experiments to examine the global gene expression changes that occur upon differentiation to the swarmer cell. We hypothesized that growth on a surface would elicit a specific response involving genes for motility and surface colonization and not the broad changes in physiology suggested by others to be co-regulated with swarming motility. By taking advantage of the two known signals required for swarmer cell induction (inhibiting polar flagellar rotation and limiting iron), the swarming response was artificially induced in liquid and used to define the set of genes associated with surface sensing by transcriptome analysis. This approach avoided the confounding physiological differences between growth in liquid and growth on a surface. Fifteen microarrays performed with different strains and growth conditions were used to define a concise set of about 70 genes that comprise the core set of surface-induced genes. This set includes genes encoding the surface motility system lateral flagella and virulence factors including a type three secretion system (T3SS1). I showed a biological consequence of the increased expression of T3SS1 genes, as surface-induced cells were more toxic in a tissue culture infection than either liquid-grown or surface-grown non-swarming mutants. I explored the role of calcium signaling in regulating the surface sensing network, as calcium seemed a pertinent signal to a marine organism and low calcium is a known inducing signal for T3SS in other organisms. Calcium was shown to enhance swarming motility and lateral flagellar gene expression. Microarrays were used to analyze the transcriptome response to growth with EGTA (a cation chelator commonly used to generate low calcium) or calcium. Surprisingly, both low and high calcium induced T3SS1 gene expression. The EGTA effect was determined to be the result of iron limitation, which was thus shown to be a new inducing signal for T3SS1. I overexpressed the master transcriptional regulator of the T3SS system, encoded by exsA, to define the entire set of T3SS1-associated genes. I found that ExsA was also a new regulator of the surface sensing regulon, which was repressed when exsA was overexpressed. Microarray analysis showed that calcium is a global regulator, controlling transcription of about 50 genes under the conditions tested. I characterized a new calcium-regulated transcription factor that we named CalR, and showed that CalR repressed swarming motility and T3SS1 gene expression. The transcription factor OpaR was previously known to repress swarming genes and control colony opacity. It is homologous to the output regulators of the quorum sensing pathway in other Vibrio species. I used microarray analysis and mutant strains to explore the functionality of the quorum sensing cascade in V. parahaemolyticus and define the OpaR regulon during growth on a surface. I showed that the quorum sensing regulator LuxO when active silences opaR as it does in other Vibrios, using a translational reporter fusion in opaR. I used microarray analysis to show that 323 genes are induced or repressed by OpaR. The surface-sensing regulon is repressed by OpaR. Many genes encoding proteins involved in virulence, signal transduction, and modulation of the signaling molecule cyclic dimeric GMP are regulated by OpaR. The quorum sensing controlled network of gene expression in V. parahaemolyticus is quite distinct from other Vibrios, with respect to both the specific nature as well as the direction of regulation of genes controlled by OpaR.

Pseudomonas aeruginosa is a Gram-negative bacterium that causes acute nosocomial infections as well as chronic infections in cystic fibrosis (CF) patients. P. aeruginosa utilizes a type III secretion system (T3SS) during acute infections to promote host cell cytotoxicity and inhibit phagocytosis. Regulation of T3SS expression can be classified into two distinct categories: intrinsic and extrinsic. T3SS intrinsic regulation involves the well-characterized ExsECDA cascade that controls T3SS gene transcription. Extrinsic regulation involves global regulatory systems that affect T3SS expression. Despite general knowledge of global regulation of T3SS expression, few specific mechanisms have been elucidated in detail. The overall goal of my thesis work was to provide clarity to global regulatory mechanisms controlling T3SS expression. One well-documented observation is that P. aeruginosa isolates from CF patients commonly have reduced T3SS expression. In chapter II, I describe how the MucA/AlgU/AlgZR system, commonly activated in CF isolates through mutation of the mucA gene, inhibits T3SS gene expression. My experiments demonstrate that the AlgZR two-component system inhibits ExsA expression through two separate global regulatory systems. First, as previously described, AlgZR inhibits ExsA expression by reducing activity of the cAMP/Vfr signaling pathway. Vfr, a homolog of Escherichia coli Crp, regulates T3SS gene expression through an unknown mechanism. Second, AlgZR alters the activity of the RsmAYZ system to specifically reduce ExsA expression. The RNA-binding protein RsmA, a homolog of E. coli CsrA, activates ExsA expression at a post-transcriptional level. Previous studies in our laboratory identified several transposon insertion mutants that appeared to be novel extrinsic regulators of T3SS gene expression. One of those candidates, named DeaD, is a putative ATP-dependent RNA helicase. My experiments in chapter III reveal that DeaD regulates T3SS expression by directly stimulating exsA translation. Mutants lacking deaD have reduced exsA translational reporter activity and ExsA expression in trans fails to complement a deaD exsA double mutant for T3SS gene expression. I demonstrate that purified DeaD stimulates ExsA expression in a coupled in vitro transcription/translation assay, confirming our in vivo findings. In chapter II, I observed that RsmA activates the transcription of RsmY and RsmZ, two small non-coding RNAs that act to sequester RsmA from target mRNAs. My experiments in chapter IV begin to dissect the RsmA-activation mechanism of RsmY/Z expression. I show that RsmA activation requires the previously described Gac/Lad/Ret system that controls RsmY/Z expression. RsmA, however, does not alter Gac/Lad/Ret gene transcription or translation. Interestingly, an RsmA variant deficient in RNA-binding, RsmA R44A, was able to complement an rsmA mutant for RsmY/Z expression. I hypothesized that RsmA interacts with an unknown protein to activate RsmY/Z expression and identified several potential interaction partners using co-purification assays. Together, my combined experiments elucidate novel global regulatory pathways controlling T3SS gene expression during acute and chronic P. aeruginosa infections, and provide a foundation towards the goal of developing future treatment options.

Transcription of the Pseudomonas aeruginosa type III secretion system is controlled by ExsA, a member of the AraC/XylS family of regulators. ExsA is comprised of an amino terminal domain that is involved in self-association and regulatory functions, and a carboxy-terminal domain that contains two helix-turn helix (HTH) DNA-binding motifs which contact promoter DNA. Previous work from our lab determined the function of the two independent ExsA domains and found that each ExsA-dependent promoter contains two adjacent binding sites for monomeric ExsA. The promoter-proximal site (binding site 1) consists of highly conserved GnC and TGnnA sequences that are individually recognized by the two HTH DNA-binding motifs of an ExsA monomer. Nevertheless, the details of how ExsA recognizes and binds to ExsA-dependent promoters were still unknown. In chapter II I show that the two ExsA monomers bind to promoter regions in a head-to-tail orientation and identify residues in the first HTH of ExsA that contact the GnC sequence. Likewise, residues located in the second HTH motif, which contribute to the recognition of the TGnnA sequence, were also identified. While the GnC and TGnnA sequences are important for binding to site 1, the promoter-distal binding sites (site 2) lack obvious similarity among themselves or with binding site 1. Site 2 in the PexsC promoter region contains a GnC sequence that is functionally equivalent to the GnC in site 1 and recognized by the first HTH motif of an ExsA monomer and the second HTH interacts with an adenine residue in binding site 2. A comparison of hybrid promoters composed of binding site 2 from one promoter fused to binding site 1 derived from another promoter indicates that ExsA-binding affinity, promoter strength, and the degree of promoter bending are properties that are largely determined by binding site 2. Through the course of the ExsA studies I observed that the amino acids that comprise the HTH motifs of ExsA are nearly identical to those in LcrF/VirF, the activators of T3SS gene expression in the pathogenic yersiniae. In chapter III I tested the hypothesis that ExsA/LcrF/VirF recognize a common nucleotide sequence. Here I report that Yersinia pestis LcrF binds to and activates transcription of ExsA-dependent promoters in P. aeruginosa, and that plasmid expressed ExsA complements a Y. pestis lcrF mutant for T3SS gene expression. Mutations that disrupt the ExsA consensus-binding sites in both P. aeruginosa and Y. pestis T3SS promoters prevent activation by ExsA and LcrF. All of the data combined demonstrate that ExsA and LcrF recognize a common nucleotide sequence. Nevertheless, the DNA binding properties of ExsA and LcrF are distinct. Whereas two ExsA monomers are sequentially recruited to the promoter region, LcrF binds to promoter DNA as a preformed dimer and has a higher capacity to bend DNA. An LcrF mutant defective for dimerization bound promoter DNA with properties similar to ExsA. Finally, I demonstrate that the activators of T3SS gene expression from Photorhabdus luminescens, Aeromonas hydrophila, and Vibrio parahaemolyticus are also sensitive to mutations that disrupt the ExsA-consensus binding site. Taken together, this work shows that ExsA binding and activation at T3SS gene promoters serves as a model system by which the DNA binding properties of other AraC family transcriptional activators can be predicted.

The costs associated with cellular product recovery commonly account for as much as 80% of the total production expense. As a specific example, significant recovery costs limit commercial use of polyhydroxyalkanoates (PHA), which comprise a class of microbially-accumulated polyesters. PHAs are biodegradable compounds that are of interest as a sustainable alternative to petrochemically-derived plastics. Secretion-based recovery of PHAs was studied to decrease PHA production costs. Type I and II secretory pathways are commonly used for the translocation of recombinant proteins out of the cytoplasm of E. coli. Proteins were targeted for translocation using four signal peptides (HlyA, TorA, GeneIII, and PelB) that operate via type I and II secretory machinery. GFP translocation was investigated in parallel due to its relative ease of monitoring to gather information about the functionality of signal peptide sequences. The translocation of phasin was investigated because of its physical binding interaction with the PHA granule surface. Genetic fusion of phasin with targeting signal peptides creates a PHA-phasin-signal peptide complex that can then be potentially used for cellular export. An important design aspect of this investigation is that synthetic biological engineering principles and standardized technical formats BBF RFC 10 and BBF RFC 23 were applied for more efficient construction of genetic devices. As an additional part of this study, an 1H NMR-based PHA quantification method was developed to facilitate analysis of intracellular PHAs. Overall, this study demonstrated that the BioBrick model can be used to construct functional devices that promote secretion of cellular compounds. The information gathered from this work can be further optimized and applied to more complex cellular manufacturing systems.

Comprendre l’émergence des maladies dans les agroécosystèmes nécessite d’étudier l’histoire évolutive des populations bactériennes associées aux plantes. L’objectif de ce travail était de déterminer les évènements évolutifsconduisant à l’émergence des lignées pathogènes ou pathovars dans l’espèce Xanthomonas arboricola. Une analyse de génétique des populations a été menée sur un panel de souches phytopathogènes et commensales et complétée par l’inférence des gains et pertes de facteurs de virulence. Cette espèce possède une structure de population épidémique ; les clones épidémiques ont émergé suite à l’acquisition de facteurs de virulence à partir d’un fond recombinant de souches commensales. Une analyse de génomique des populations et la reconstruction de scénarios de divergence entre ces clones et le réseau de souches recombinantes, a montré la persistance d’un flux de gènes asymétrique entre ces deux groupes, dans le sens souches pathogènes vers souches commensales. Enfin, l’histoire évolutive du principal facteur de virulence des Xanthomonas, le système de sécrétion de type 3, a été retracée au sein du genre, et a montré que celui-ci avait été acquis ancestralement puis perdu dans certaines souches commensales. En conclusion, l’ancêtre commun de X. arboricola possédait des facteurs de virulence et au sein des souches commensales, certaines ont perdu ces facteurs, tandis que d’autres ont conservé le répertoire ancestral. Ces dernières diffèrent peu de certains agents pathogènes, et pourraient représenter un risque pour de nouvelles émergences. Des travaux de génomique fonctionnelle permettraient de valider ces hypothèses. / Deciphering the evolutionary history of bacterial populations associated to plants is necessary to understand diseaseemergence in agroecosystems. The aim of this study is to unveil the evolutionary events responsible for pathogeniclineages or pathovar emergences in Xanthomonas arboricola. This species is composed of both plant pathogenic andcommensal strains Population genetics analyses and gain and loss inferences of virulence factors showed that X. arboricola exhibits an epidemic population structure, within which epidemic clones emerged from a recombinogenic background population following virulence factor acquisition. Population genomics and inference of divergence scenarii between epidemic clones and the network of recombinant strains showed persistence of homologous recombination along divergence of these two groups, with an asymmetric gene flux from pathogenic strains to commensal ones. Finally, evolutionary history of the type three secretion system (T3SS), the main virulence factor in Xanthomonas genus, was studied at genus scale and showed that T3SS was ancestrally acquired and lost in commensal strains. Altogether these analyses allowed us to show that the common ancestor of X.arboricola had virulence factors, and that within commensal strains, some lost these virulence factors whereas others kept the ancestral repertoire. These latter strains have a similar repertoire to that of some pathogenic strains, and could represent a risk for new disease emergence. Functional genomics could allow us to validate these hypotheses.