Environmental signals are sensed by membrane-spanning receptors that communicate with the cell interior. Bacterial chemoreceptors modulate the activity of the CheA kinase in response to binding of small ligands or upon interaction with substrate-bound periplasmic-binding proteins. The mechanism of signal transduction across the membrane is a displacement of the second transmembrane domain (TM2) a few angstroms toward the cytoplasm. This movement repositions a dynamic transmembrane helix relative to the plane of the cell membrane. The research presented in this dissertation investigated the contribution of TM2-membrane interactions to signaling by the aspartate chemoreceptor (Tar) of Escherichia coli. Aromatic residues that reside at the cytoplasmic polar-hydrophobic membrane interface (Trp-209 and Tyr-210) were found to play a significant role in regulating signaling by Tar. These interactions were subsequently manipulated to modulate the signaling properties of Tar. The baseline signaling state was shown to be incrementally altered by repositioning the Trp-209/Tyr-210 pair. To our knowledge, this is the first example of harnessing membrane-protein interactions to modulate the signal output of a transmembrane receptor in a controlled and predictable manner. Potential long-term applications include the use of analogous mutations to elucidate two-component signaling pathways, to engineer the signaling parameters of biosensors that incorporate chemoreceptors, and to predict the movement of dynamic transmembrane helices in silico.

Environmental signals are sensed by membrane-spanning receptors that communicate with the cell interior. Bacterial chemoreceptors modulate the activity of the CheA kinase in response to binding of small ligands or upon interaction with substrate-bound periplasmic-binding proteins. The mechanism of signal transduction across the membrane is a displacement of the second transmembrane domain (TM2) a few angstroms toward the cytoplasm. This movement repositions a dynamic transmembrane helix relative to the plane of the cell membrane. The research presented in this dissertation investigated the contribution of TM2-membrane interactions to signaling by the aspartate chemoreceptor (Tar) of Escherichia coli. Aromatic residues that reside at the cytoplasmic polar-hydrophobic membrane interface (Trp-209 and Tyr-210) were found to play a significant role in regulating signaling by Tar. These interactions were subsequently manipulated to modulate the signaling properties of Tar. The baseline signaling state was shown to be incrementally altered by repositioning the Trp-209/Tyr-210 pair. To our knowledge, this is the first example of harnessing membrane-protein interactions to modulate the signal output of a transmembrane receptor in a controlled and predictable manner. Potential long-term applications include the use of analogous mutations to elucidate two-component signaling pathways, to engineer the signaling parameters of biosensors that incorporate chemoreceptors, and to predict the movement of dynamic transmembrane helices in silico.

No description available.

Pseudomonas aeruginosa is an important nosocomial infection that has the potential to infect almost every tissue of the human body though it is mainly opportunistic, due to the organism’s intrinsic antibiotic resistance is becoming increasingly difficult to treat. Two-component systems (TCS) rely on a signal sensed from the outside environment by the sensor histidine kinase to initiate phosphotransfer to the response regulator, which may then regulate virulence factors in the organism in response to a changing environment. One important two-component system in P. aeruginosa is the AlgZ/R system. AlgZ, the sensor histidine kinase, has been shown to be co-transcribed with its’ response regulator, AlgR, to affect a myriad of virulence factors, including those related to motility. Pseudomonas species is capable of four types of motility: twitching, swimming, swarming, and gliding. Twitching motility is achieved through the expression of the FimU operon and Type VI pilli, and is most useful when attaching to a solid surface in the initial step of pathogenesis: colonization. Conversely, the swimming phenotype relies on the production of a single polar flagellum upon the activation of the FleQ operon, and allows the organism to move through a fluid environment. A previously unidentified regulator of AlgZ, but not AlgR, has been identified via transposon mutagenesis screening, PA2771, which has a GGDEF domain and predicted diguanylate cyclase activity. The mechanism of PA2771’s action within P. aeruginosa has not been previously studied. The nonpolar deletion mutant was first characterized via various phenotypic assays (including biofilm, rhamnolipid, swimming, and swarming assays) and transcriptional fusions to propose a mechanism in which this predicted diguanylate cyclase (DGC) works with AlgZ to determine the switch in motility from twitching to swimming. When PA2771 is present and active in the cell, cyclic di-GMP levels should be high, leading to the production of Type VI pilli and the upregulation of the FimU operon. In the PA2771 mutant a significant decrease in the expression of the FimU operon, and an increase in the expression of the flagellar genes. Subsequent alterations in swimming and swarming phenotypes were observed, as well as the restoration of these effects via complementation studies. Overexpression of AlgZ in the 2771 mutant showed a restoration of AlgZ expression in the nonmucoid background, and the predicted DGC activity was indirectly verified via a cdrA-lacZ transcriptional fusion.

Signal transduction networks enable organisms to respond to environmental stimuli. Bacteria utilize two-component systems (TCSs) and phosphorelays as their primary means of signal transduction. Histidine kinase (HK) and response regulator (RR) proteins comprise these TCSs and phosphorelays. Previously, signal transduction within TCSs and phosphorelays was thought to only occur through a linear series of phosphotransfers between HKs and RRs. Recently multikinase networks have been shown to be involved in TCS and phosphorelay signal transmission. A multikinase network that includes the HKs RetS and GacS controls the switch between the motile invasive lifestyle and the sessile biofilm lifestyle of the opportunistic human pathogen Pseudomonas aeruginosa. GacS promotes the sessile biofilm lifestyle, while RetS promotes the motile invasive lifestyle via the inhibition of GacS. This inhibition occurs through three distinct mechanisms. Two of the mechanisms are dephosphorylating mechanisms and the third mechanism is a direct interaction between RetS and GacS which results in the inhibition of GacS autophosphorylation. This study examines the direct binding interaction between RetS and GacS using structural biology. We observed a heterodimeric RetS-GacS complex in which the canonical homodimerization interface was replaced with a heterodimeric interface. Heterodimerization between bacterial HKs is currently a novel observation, but it is likely that other HKs heterodimerize. The RetS-GacS direct interaction can serve as a model for HK-HK binding in multikinase networks. / Doctor of Philosophy / The way in which bacteria assess and respond to their environment is of great interest to microbiologists. Bacteria transmit environmental signals via protein interactions. Some of these interactions involve the transfer of phosphate groups, and some involve a direct binding interaction between proteins. We are investigating a direct binding interaction between two proteins, RetS and GacS. These proteins control whether Pseudomonas aeruginosa, an opportunistic pathogen of humans, causes an acute infection, which is characterized by motility and invasiveness, or a chronic infection, which is characterized by a sessile biofilm lifestyle, in a human host. Through the use of structural biology techniques we have visualized the three-dimensional structure of the complex between RetS and GacS. This complex has provided insight into the role of the RetS-GacS interaction in controlling the infection state of P. aeruginosa.

Part I: Signaling Role of the Sensor Kinase RetS in Biofilm formation Regulation of Pseudomonas aeruginosa-<br />The opportunistic human pathogen Pseudomonas aeruginosa causes both acute and chronic infections in predisposed individuals. Acute infections require a functional Type Three Secretion System (TTSS), which mediates the translocation of select cytotoxins into host cells. Chronic infections, the leading cause of death among cystic fibrosis patients, are characterized by drug-resistant biofilms formation. To regulate gene expression, Pseudomonas aeruginosa utilizes two-component regulatory systems (TCS). Specifically, we focus on the TCS signaling kinase RetS, which is a critical repressor of biofilm formation. The signaling mechanism of RetS is unusual. According to recent findings and one hypothesis, RetS employs a novel signaling mechanism involving direct binding to the signaling kinase GacS, thereby repressing the GacS-induced biofilm formation. RetS is believed to be regulated by the interaction of its periplasmic sensory domain (RetSperi) with an unknown ligand. As such, RetSperi is a potential drug target. We hypothesized that ligand-binding shifts the equilibrium between the formation of a RetS homo-dimer and the RetS-GacS complex by tuning the homo-dimerization of the RetSperi. While the molecular signal that regulates RetS is unknown, our structural studies of the sensory domain suggest that this ligand is a carbohydrate-based moiety. Unchanged biofilm-EPS production phenotype of RetSperi ligand binding site mutants indicates that the natural ligand is not from Pseudomonas aeruginosa.<br />Additional experiments unambiguously determined that the sensory domain forms a stable homodimer. Adding to the complexity of the system, we have identified<br />two possible dimer interfaces in our in vitro assays. However, inconsistent with the current model, elimination of RetSperi results in a slightly increased biofilm EPS production phenotype. Therefore, with the previous demonstration that RetS is able to dephosphorylate GacS, we propose an alternative hypothesis: the RetS kinase domain serves as a phosphatase for phosphorylated GacS; this phosphatase activity is tuned by signaling sensing on RetSperi. Finally, to provide an important piece of information for understanding the molecular basis of RetS-GacS signaling, we have developed a crystallization-based structure determination strategy in order to reveal the precise RetS-GacS interaction pattern.<br /><br />PartII: The catalytic domain of the germination-specific lytic transglycosylase SleB from Bacillus anthracis displays a unique active site topology-<br />germination-specific lytic enzymes (GSLEs) that degrade the unique cortex peptidoglycan to permit resumption of metabolic activity and outgrowth. We report the first crystal structure of the catalytic domain of a GSLE, SleB. The structure revealed a transglycosylase fold with unique active site topology and permitted identification of the catalytic glutamate residue. Moreover, the structure provided insights into the molecular basis for the specificity of the enzyme for muramic-"?lactam-containing cortex peptidoglycan. The protein also contains a metal-binding site that is positioned directly at the entrance of the substrate-binding cleft. / Ph. D.

Tous les organismes, les animaux, les plantes et les microbes, sont composés de cellules polarisées, en affichant un positionnement asymétrique des organites sub-cellulaires ou des structures. Le contrôle de polarité a été étudié chez les eucaryotes pendant une longue période, et a été montré pour e&#770;tre impliqués dans de nombreux processus physiologiques, tels que l'embryogenèse, le cancer métastatique et les maladies dégénératives des neurones. Chez les procaryotes, des études de polarité ne sont apparues récemment avec le développement de la microscopie à fluorescence sensibles. Ces études ont révélé que les cellules procaryotes sont en fait très organisé et une masse croissante de la littérature a montré que les cellules bactériennes également utiliser des radeaux lipidiques, courbure membranaire, la paroi cellulaire et un cytosquelette complexe pour diriger le positionnement spécifique de structures subcellulaires.Petites GTPases de la superfamille Ras sont des éléments réglementaires polarisation répandue chez les eucaryotes. Malgré l'existence depuis longtemps de ces petites GTPases dans les génomes procaryotes, leur fonction a jamais été étudiée. Pendant ce travail de thèse, nous avons trouvé, pour la première fois, qu'une petite GTPase, MglA et de sa protéine apparentée Activation GTPase (GAP) MglB, directe une dynamique axe antéro-postérieur à la motilité directe en forme de tige deltaproteobacterium Myxococcus xanthus. Dans ce processus, MglA s'accumule dans son état lié au GTP au niveau du po&#770;le leader de cellules, en activant les machineries motilité. Ce schéma de localisation est maintenue par MglB, qui localise le pôle opposé, le blocage de l'accumulation MglA à ce po&#770;le à travers son activité GAP. Remarquablement, les deux protéines passer leur localisation synchrone, ce qui correspond à un changement dramatique dans la direction du mouvement cellulaire (inversion). Ce commutateur est réglementé par un système chimiosensoriels-like, Frz. Dans une deuxième partie de ce travail, nous avons identifié un régulateur de protéine de réponse, RomR qui est essentiel pour le regroupement polaire de MglA. Interdépendances complexes entre la localisation RomR, MglA et MglB indiquent que ces protéines pourraient constituer un complexe de polarité dynamique de trois protéines qui rec&#807;oit Frz de signalisation pour passer l'axe de polarité. En conclusion, les résultats de ce travail de thèse suggère que M. xanthus intégré un module de polarité eucaryotes-like (MglAB) dans un procaryote spécifique (Frz) réseau de signalisation pour réguler sa motilité. Une telle réglementation est distincte sous forme de petites protéines G des règlements, qui sont généralement couplés à la protéine G récepteurs couplés (GPCR) chez les eucaryotes. Enfin, ce travail ouvre la voie pour comprendre comment la réglementation seule la motilité cellulaire sont intégrés pour générer des comportements commandés multicellulaires donnant naissance à des structures primitives de développement, par exemple, la morphogenèse du corps fructifères. D'autre part, ce travail fournit également un exemple d'analyser les étapes évolutives donnant lieu à des réseaux de signalisation. / All organisms, animals, plants and microbes, are composed of polarized cells, displaying asymmetric positioning of sub-cellular organelles or structures. Polarity control has been studied in eukaryotes for a long time, and has been shown to be involved in many physiological processes, such as embryogenesis, cancer metastasis and neuron degenerative diseases. In prokaryotes, polarity studies only emerged recently with the development of sensitive fluorescent microscopy. These studies revealed that prokaryotic cells are in fact highly organized and a growing body of literature has shown that bacterial cells also use lipid rafts, membrane curvature, the cell wall and a complex cytoskeleton to direct the specific positioning of subcellular structures.Small GTPases of the Ras superfamily are widespread polarization regulatory elements in eukaryotes. Despite the long known existence of such small GTPases in prokaryotic genomes, their function has never been studied. During this thesis work, we found, for the first time, that a small GTPase, MglA and its cognate GTPase Activating Protein (GAP) MglB, direct a dynamic anterior- posterior axis to direct motility of the rod-shaped deltaproteobacterium Myxococcus xanthus. In this process, MglA accumulates in its GTP-bound state at the leading cell pole, activating the motility machineries. This localization pattern is maintained by MglB, which localizes at the opposite pole, blocking MglA accumulation at this pole through its GAP activity. Remarkably, both proteins switch their localization synchronously, which correlates with a dramatic change in the direction of cell movement (reversal). This switch is regulated by a chemosensory-like system, Frz. In a second part of this work, we identified a response regulator protein, RomR which is essential for the polar clustering of MglA. Intricate localization interdependencies between Romr, MglA and MglB indicate that these proteins might constitute a dynamic three-protein polarity complex that receives Frz-signaling to switch the polarity axis. In conclusion, the results from this thesis work suggest that M. xanthus integrated a eukaryotic-like polarity module (MglAB) into a prokaryotic- specific (Frz) signaling network to regulate its motility. Such regulation is distinct form small G- protein regulations, which are generally coupled to G-protein coupled receptors (GPCRs) in eukaryotes. Finally, this work paves the way to understand how single cell motility regulations are integrated to generate ordered multicellular behaviors giving rise to primitive developmental structures, for example fruiting body morphogenesis. On the other hand, this work also provides an example to analyze the evolutionary steps giving rise to signaling networks.

Mycobacterium tuberculosis, the causative organism of the disease tuberculosis (TB) in humans, leads to nearly two million deaths each year. This versatile pathogen can exist in highly distinct physiological states such as asymptomatic latent TB infection where bacilli lie dormant or as active TB disease in which the bacilli replicate in macrophages. The pathogenic lifestyle requires the tubercle bacillus to sense and respond to multiple environmental cues to ensure its survival. Such stimuli include hypoxia, nutrient limitation, presence of reactive oxygen and reactive nitrogen intermediates, pH alterations, and cell wall/ membrane stress. Two component systems (TCSs) form the primary apparatus for sensing and responding to environmental cues in bacteria. A prototypical TCS is composed of a sensory protein called sensor kinase (SK) and a response generating protein called response regulator (RR). M. tuberculosis encodes 11 genetically paired TCSs, 2 orphan sensor kinases and six orphan response regulator proteins. Studies of the TB bacilli using transcriptional profiling and gene knockouts have revealed that TCSs play an important role in facilitating successful adaptation to diverse environmental conditions encountered within the host. The mtrAB and prrAB genes encoding corresponding TCSs have been shown to be essential for survival, mprAB for persistence and devRS for hypoxic adaptation. Further, inactivation of the TCSs regX3-senX3, tcrXY, trcRS, phoPR or kdpDE was shown to affect the growth and/or virulence of M. tuberculosis in animal infection models. The SK and RR proteins of TCSs are modular and contain variable input and output domains coupled to conserved ‘transmitter’ and ‘receiver’ domains. Despite the modular nature and extensive homology of SK and RR proteins across TCSs, which may allow non-cognate interactions, it is believed that crosstalk across different TCSs is not favored and that individual pathways are generally well insulated. The existing profiling studies have been performed on the TCSs of bacterial species containing a relatively large number of TCSs. In those studies, specificity and insulation have been the norm and thus become the prevalent paradigm of TCS signaling. In vitro genome wide phosphotransfer profiling has revealed only a few cross- communication nodes in the TCSs of Escherichia coli (~3%), while none in Caulobacter crescentus (in 352 interactions tested, in short time duration) and Myxococcus xanthus (in 250 interactions tested). Yet, many instances of cross talk have been reported in literature. For example, E. coli TCSs PmrAB and EnvZ-OmpR show cross-communication with QseBC and ArcBA, and many more. In M. tuberculosis, indirect evidence of the existence of such cross regulation has originated from studies where mutations in phoPR have been shown to affect the expression of the TCS devRS and its regulon. It is thus interesting to examine the extent of crosstalk in the TCSs of M. tuberculosis, which has an exceptionally small number of TCS proteins compared to E. coli. As mentioned earlier, M. tuberculosis H37Rv has 11 cognate pairs of TCSs, 2 orphan sensor kinases and 6 orphan response regulators. To study the entire landscape, we aimed to study all 221 connections between SK and RR proteins including 12 cognate interactions. While 10 of the cognate TCS interactions were established in the literature, two putative systems KdpDE and NarSL and 5 orphan response regulators were still uncharacterized, therefore we initiated our work with the characterization of these TCSs. At the biochemical level, the KdpDE two component system of M. tuberculosis is not well studied, though one report showed interaction of the C-terminal domain of KdpD SK and KdpE RR using yeast two hybrid assay and another reported the interaction of the SK with LRP protein. Besides these associations, there is no evidence for the functionality of KdpDE system. Similarly, NarSL system also has not been characterized and it not known whether these putative two component proteins are functional. The initial part of the study includes the characterization of these two TCSs, NarS-NarL and KdpD-KdpE, at biochemical and physiological levels. In our studies we demonstrated that KdpDE system is a bonafide two component system of M. tuberculosis, and KdpD SK undergoes autophosphorylation at His642 residue in presence of Mg+2 ions and then it transfers phosphoryl group to a conserved Asp52 residue on the KdpE RR protein. The acid-base stability analysis revealed the nature of chemical bonds present in the KdpD and KdpE proteins, and further confirmed that KdpD and KdpE are typical SK and RR respectively. SPR analysis demonstrated that KdpD and KdpE proteins interact under basal non-phosphorylated conditions and the interaction affinity reduced when SK was phosphorylated. The reduction in the interaction affinity indicated towards a possible dissociation of SK and RR protein during phosphotransfer, which allows RRs to exert their regulatory effect. On the similar line, the phosphorylation defective SK (KdpDH642Q) had least affinity with KdpE suggesting that perhaps this mutant SK, fails to interact with the RR. We have also shown that both the kdpD and kdpE genes are in the same operon and are up regulated in potassium ions limitation and osmotic stress conditions. Overall, using the biochemical approaches, we have established that Rv1027c–Rv1028c operon of M. tuberculosis encodes a functional and a typical KdpDE two component signal transduction system. Using the similar biochemical and biophysical approaches, we have demonstrated that NarS-NarL proteins constitute a functional TCS and His241 and Asp61 are the phosphorylatable residues. In contrast to KdpDE which shows typical behaviour of TCS, NarSL TCS showed atypical behaviour. Malhotra and group’s work on NarSL suggested that there is cross-regulation between NarS/NarL and DevS/DosT/DevR systems. We addressed this possibility on three separate levels, by examining (i) the cross-phosphorylation of DevR and NarL RRs by non-cognate sensor kinases NarS and DevS/DosT respectively, (ii) the interaction between DevR and NarL RR proteins, and (iii) examining the effect of DevR-NarL interactions on their DNA binding properties. Our studies ruled out the presence of any physiologically relevant phosphorylation mediated cross-talk between NarS/NarL and DevS/DosT/DevR. We identified that the cross talk between these TCSs could be explained on the basis of interaction between NarL and DevR RRs and their subsequent binding to the target gene promoter regions for concerted regulation of gene expression. We also identified that DevR activation is critical for cooperative action with NarL. This process comes out as a novel mechanism of gene regulation via heteromerization of RRs. We hypothesized that formation of NarL-DevR heteromers may arise because of high sequence similarities. Conclusively, our study provides insights into the functionality of M. tuberculosis NarL/NarS TCS and regulatory function of NarL protein which acts in concert with another RR, DevR. Overall, NarS-NarL system showed an atypical, novel mode of gene regulation involving RR heteromerization. Subsequent to the basic biochemical characterization of NarSL and KdpDE system, the genome wide phosphotransfer profiling was done to identify the cross-connections between TCSs. Remarkably, we found that specificity was the exception rather than the rule. While only three of the TCS pairs were completely specific, all the other nine TCS pairs exhibited crosstalk, including a few that were highly promiscuous. We classified the interactions as specific, one-to-many, and many-to-one signaling circuits. We also profiled all the RRs including the orphans for their ability to accept phosphoryl group from a low molecular weight donor, acetyl phosphate, and interestingly found that only two RRs DevR and NarL were capable of accepting phosphoryl group from such a donor. Interestingly, none of the orphan RRs accepted phosphoryl group from any donor, neither SKs nor low molecular weight phospho donors, warranting further analysis of their roles and presence in the M. tuberculosis genome. Our exhaustive map of the crosstalk between the TCSs of M. tuberculosis sets the stage for a renewed view of TCS signaling and proposes a dispersive-integrative landscape for TCS signaling rather than one of insulation. As an extension of our basic characterization work of NarSL TCS, we also attempted to understand the localization pattern of NarS sensor kinase in M. smegmatis cells using fluorescence approaches. It is known that many bacterial receptors including sensor kinases form clusters or show specific localization patterns inside the cell. We found that NarS shows distinct cellular localization pattern. However, the functional significance of this localization pattern is not obvious yet and warrants further investigations. We also developed a few non-radioactive methods to study interaction between two component systems to overcome the limitations associated with radioactive experiments in studying TCSs. We developed fluorescence resonance energy transfer (FRET) to study in vitro interaction between two component proteins which was sensitive to the phosphorylation status of the proteins. Using fluorescently tagged SKs and RRs, we determined a change in FRET for KdpDE and NarSL TCS pairs in vitro. Our study thus also provides an alternative approach to study TCS signaling, using an easier, non-radioactive and high throughput approach. In summary, our study presents the evidence of an alternative paradigm of bacterial signaling, where significant crosstalk between the underlying TCSs prevails. The new paradigm is expected to have important implications in our understanding of the virulence and pathogenesis of bacterial infections. Overall, our studies (i) allowed the establishment of functionality of all paired TCSs encoded in the genome of M. tuberculosis including NarSL and KdpDE TCSs, (ii) identified the novel mechanism of gene regulation by NarL RR and DevR, (iii) demonstrated the existence of TCS signaling which is contrary to the existing notion of specificity (iv) showed the distinct localization pattern of NarS and (v) developed non-radioactive approaches to study two component interactions.

La résistance aux polymyxines chez P. aeruginosa résulte en partie de la modification du lipide A par addition de 4-amino-aL-arabinose (L-Ara4N), due à l'expression de l'opéron arnBCADTEF-ugD (arn), activée par au moins 6 systèmes de régulation à deux composants (S2C). Nous avons mis en évidence que P. aeruginosa était capable de s'adapter de manière transitoire à la présence de fortes concentrations de polymyxines (8 x CMI) aussi bien in vitro que in vivo dans un modèle murin d'infection pulmonaire aiguë. La délétion de l'opéron arn chez la souche sauvage n'a pas modifié la capacité d'adaptation de P. aeruginosa. Afin d'identifier les gènes impliqués dans le processus adaptatif, le transcriptome global du mutant PAOl/z«;-Aar«-ATCS délété des principaux S2C (parRS, pmrAB, cprRS eiphoPQ) et de l'opéron arn a été réalisé en présence de différentes concentrations de colistine par RNA-Seq. Deux nouveaux mécanismes ont ainsi été identifiés. L'un repose sur l'expression de l'opéron mmsAB codant des enzymes du catabolisme des acides gras et l'autre fait intervenir le facteur sigma alternatif AlgU. Seule la délétion conjointe du gène algW participant à l'activation de AlgU et des opérons arn, mmsAB, pmrAB, parRS, phoPQ et cprRS a permis d'abolir complètement la résistance adaptative à la colistine. Par ailleurs, nous avons mis en évidence le rôle des vésicules de membrane externe (OMVs) dans la séquestration de l'antibiotique, dont la production semble régulée au moins par AlgU et le PQS. Ces travaux offrent des perspectives intéressantes pour l'identification de nouvelles cibles antibactériennes et pour l'amélioration de l'effet bactéricide des polymyxines. / Resistance to polymyxins in Pseudomonas aeruginosa involves the addition of 4-amino-L-arabinose (Ara4N) to LPS phosphates, thanks to an enzymatic modification due to the operon named arnBCADTEF-ugD (arn) whose expression is activated by at least 6 two component regulatory Systems (TCS). We demonstrated that P. aeruginosa was able to resist in a transient way to high concentrations of polymyxins (8x MIC) in vitro and in vivo in a mice lung infection model. Arn operon deletion in the wild type strain did not modify the ability to adapt to polymyxins. In order to identify gènes involved in adaptive resistance, we performed RNA-seq transcriptomes of quintuple mutant PAO\lux-Aaw-ATCS exposed to different concentrations of colistin or non exposed. Two new mechanisms were identified. The first one is based upon mmsAB operon encoding fatty acid catabolism enzymes and the second one is due to the sigma factor AlgU. Only the deletions of algW%enz involved in AlgU activation and arn, mmsAB, pmrAB, parRS, phoPQ and cprRS operons completely abolished the adaptive process. We also demonstrated the role of outer membrane vesicles in the sequestration of colistin whose production is regulated by AlgU and PQS. This study provides knoweldge essential for the design of novel strategies aimed at tackling the adaptive resistance to polymyxins.

Pseudomonas aeruginosa is a Gram-negative, metabolically versatile, opportunistic pathogen that exhibits a multitude of virulence factors, and is extraordinarily resistant to a gamut of clinically significant antibiotics. This ability is in part mediated by two-component systems (TCS) that play a crucial role in regulating virulence mechanisms, metabolism and antibiotic resistance. Our sequence analysis of the P. aeruginosa PAO1 genome revealed the presence of two open reading frames, mifS and mifR, which encodes putative TCS proteins, a histidine sensor kinase MifS and a response regulator MifR, respectively. This two-gene operon was found immediately upstream of the poxAB operon, where poxB encodes a chromosomal ß-lactamase, hinting at the role of MifSR TCS in regulating antibiotic resistance. However, loss of mifSR had no effect on the antibiotic resistance profile when compared to P. aeruginosa parent PAO1 strain. Subsequently, our phenotypic microarray data (BioLOG) and growth profile studies indicated the inability of mifSR mutants to grow in α-ketoglutarate (α-KG), a key tricarboxylic acid (TCA) cycle intermediate, as a sole carbon source. To date, very little is known about the physiology of P. aeruginosa when provided with α-KG as its sole carbon source and the role of MifS and MifR TCS in virulence. Importantly, in the recent years, α-KG has gained notoriety for its newly identified role as a signaling molecule in addition to its conventional role in metabolism. This led us to hypothesize that MifSR TCS is involved in α-KG utilization and virulence in P. aeruginosa. Using mifS, mifR and mifSR clean in-frame deletion strains, our study demonstrates that the MifSR TCS modulates the expression P. aeruginosa kgtP (PA5530) and pcaT (PA0229) genes encoding putative α-KG permeases. In addition, our study shows that the MifSR-regulation of these transporters requires functional sigma factor RpoN (σ54). Loss of mifSR in the presence of α-KG, resulted in differential regulation of P. aeruginosa key virulence determinants including biofilm formation, motility, cell cytoxicity and the production of pyocyanin and pyoverdine. Involvement of multiple regulators and transporters suggests the presence of an intricate circuitry in the transport of α-KG and its importance in P. aeruginosa survival. This is further supported by the α-KG-dependent MifSR regulation of multiple virulence mechanisms. Simultaneous regulation of multiple mechanisms involved in P. aeruginosa pathogenesis suggests a complex mechanism of MifSR action. Understanding the physiological cues and regulation would provide a better stratagem to fight often indomitable P. aeruginosa infections.