Tetrahydrodipicolinate N-Succinyltransferase and Dihydrodipicolinate Synthase from Pseudomonas aeruginosa: Structure Analysis and Gene Deletion

The diaminopimelic acid pathway of lysine biosynthesis has been suggested to provide attractive targets for the development of novel antibacterial drugs. Here we report the characterization of two enzymes from this pathway in the human pathogen Pseudomonas aeruginosa, utilizing structural biology, biochemistry and genetics. We show that tetrahydrodipicolinate N-succinyltransferase (DapD) from P. aeruginosa is specific for the L-stereoisomer of the amino substrate L-2-aminopimelate, and its D-enantiomer acts as a weak inhibitor. The crystal structures of this enzyme with L-2-aminopimelate and D-2-aminopimelate, respectively, reveal that both compounds bind at the same site of the enzyme. Comparison of the binding interactions of these ligands in the enzyme active site suggests misalignment of the amino group of D-2-aminopimelate for nucleophilic attack on the succinate moiety of the co-substrate succinyl-CoA as the structural basis of specificity and inhibition. P. aeruginosa mutants where the dapA gene had been deleted were viable and able to grow in a mouse lung infection model, suggesting that DapA is not an optimal target for drug development against this organism. Structure-based sequence alignments, based on the DapA crystal structure determined to 1.6 Å resolution revealed the presence of two homologues, PA0223 and PA4188, in P. aeruginosa that could substitute for DapA in the P. aeruginosa PAO1ΔdapA mutant. In vitro experiments using recombinant PA0223 protein could however not detect any DapA activity

Novel drug targets in cell wall biosynthesis exploited by gene disruption in Pseudomonas aeruginosa

This study demonstrates that cell wall targets contribute significantly to intracellular survival, in vivo growth, and pathogenesis of P. aeruginosa. In conclusion, these findings establish a link between cell wall targets and virulence of P. aeruginosa and thus may lead to development of novel drugs for the treatment of P. aeruginosa infection

Ansatz zur Etablierung von konditional/reversiblen knock-out Mäusen für das zellspezifische T-Zell Onkogen SCL

Ziel der vorliegenden Arbeit war es, ein innovatives System zur Herstellung einer konditional/reversiblen SCL-knock-out Maus zu etablieren. Es wurde deshalb ein neuer knock-in/knock-out Ansatz mit Hilfe des Tet-Systems gewählt. Im Rahmen dieser Arbeit war es möglich für die Generierung der konditionalen SCL-knock-out Maus essentielle Effektor- und Respondermauslinien zu etablieren und zu charakterisieren. Durch knock-in der rtTA-cDNA in den SCL-Lokus wurde eine Effektormauslinie hergestellt, welche in allen untersuchten hämatopoietischen Geweben rtTA exprimierte.Die zur Generierung der SCL-knock-out Maus notwendigen transgenen Responderlinien wurden ebenfalls generiert. Diese Linien wurden in einem Funktionsassay auf ihre Regulierbarkeit rekombinanter SCL-Expression untersucht. Dabei wurde eine Linie identifiziert, welche stringent/exogen regulierbar war.Effektor- und Responderlinie konnten etabliert und durch adäquate Kreuzung eine heterozygote Effektor/Responderlinie generiert werden. Die Rückkreuzung dieser Linie bei gleichzeitiger Gabe von DOX sollte in der konditionalen/reversiblen SCL-knock-out Maus resultieren. Bei Ende dieser Arbeit wurde allerdings in keinem Fall ein Rescue des letalen SCL Nullhintergrunds durch die rtTA-vermittelte rekombinante SCL-Expression beobachtet. Die in dieser Arbeit bereits hergestellten und verbesserten neuen Effektorkonstrukte sowie das Zurückgreifen auf alle vorhandenen SCL- Respondermäuse können in Zukunft zur erfolgreichen Etablierung eines exogen regulierbaren konditional/reversiblen SCL knock-out führen.The aim of this work was to establish, for the first time, conditional/reversible knock-out mice for the hematopoietic transcription factor SCL. As the ablation of SCL leads to an embryonic lethal phenotype, a new strategy using the Tet-System was applied and evaluated. Using a knock-in approach, rtTA was put under the control of SCL regulatory elements (effector). Subsequently, appropriate expression of rtTA was confirmed in spleen and peripheral blood. In addition, ten transgenic responder lines were generated carrying the SCL cDNA under the control of a tet-inducible minimal promoter (responder). All transgenic responder lines were monitored for their ability to express SCL in a tet-dependent fashion. In these experiments one line exhibited stringent controlled regulation of tet-inducible SCL. Both mouse lines were used to generate heterozygote effector/responder offspring. It was expected that adequate backcrossing would result in a conditional/reversible SCL knock-out phenotype. However, in these crosses no rescue of the lethal SCL null phenotype was observed. The novel engineered effector constructs presented in this work together with the existing responder lines may lead to the establishment of exogenously inducible and also reversible SCL null mice

Novel drug targets in cell wall biosynthesis exploited by gene disruption in Pseudomonas aeruginosa

This study demonstrates that cell wall targets contribute significantly to intracellular survival, in vivo growth, and pathogenesis of P. aeruginosa. In conclusion, these findings establish a link between cell wall targets and virulence of P. aeruginosa and thus may lead to development of novel drugs for the treatment of P. aeruginosa infection

Novel drug targets in cell wall biosynthesis exploited by gene disruption in Pseudomonas aeruginosa.

For clinicians, Pseudomonas aeruginosa is a nightmare pathogen that is one of the top three causes of opportunistic human infections. Therapy of P. aeruginosa infections is complicated due to its natural high intrinsic resistance to antibiotics. Active efflux and decreased uptake of drugs due to cell wall/membrane permeability appear to be important issues in the acquired antibiotic tolerance mechanisms. Bacterial cell wall biosynthesis enzymes have been shown to be essential for pathogenicity of Gram-negative bacteria. However, the role of these targets in virulence has not been identified in P. aeruginosa. Here, we report knockout (k.o) mutants of six cell wall biosynthesis targets (murA, PA4450; murD, PA4414; murF, PA4416; ppiB, PA1793; rmlA, PA5163; waaA, PA4988) in P. aeruginosa PAO1, and characterized these in order to find out whether these genes and their products contribute to pathogenicity and virulence of P. aeruginosa. Except waaA k.o, deletion of cell wall biosynthesis targets significantly reduced growth rate in minimal medium compared to the parent strain. The k.o mutants showed exciting changes in cell morphology and colonial architectures. Remarkably, ΔmurF cells became grossly enlarged. Moreover, the mutants were also attenuated in vivo in a mouse infection model except ΔmurF and ΔwaaA and proved to be more sensitive to macrophage-mediated killing than the wild-type strain. Interestingly, the deletion of the murA gene resulted in loss of virulence activity in mice, and the virulence was restored in a plant model by unknown mechanism. This study demonstrates that cell wall targets contribute significantly to intracellular survival, in vivo growth, and pathogenesis of P. aeruginosa. In conclusion, these findings establish a link between cell wall targets and virulence of P. aeruginosa and thus may lead to development of novel drugs for the treatment of P. aeruginosa infection

The structure of <i>P. aeruginosa</i> DapA.

<p>A. Size exclusion chromatography elution profile of <i>Pa</i>DapA (PA1010) indicating that a single species exists in solution. Based on the calibration curve (insert) the calculated molecular mass is 60 kDa. B. <i>Pa</i>DapA (PA1010) purified sample analyzed in native polyacrylamide gel electrophoresis indicating a single species. C. Stereo view of the active site of <i>Pa</i>DapA located in the center of the α/β barrel. Amino acid side chains forming the active site are indicated as stick models. Residues conserved in the three homologues PA1010, PA0223 and PA4188 are shown in yellow, while the variable positions Thr44, Arg138 and Lys109 are indicated in purple. D. Sequence conservation in DapA enzymes from <i>Escherichia coli</i>, <i>Bacillus anthracis</i>, <i>Pseudomonas aeruginosa</i> and the proposed DapA paralogues in the PAO1 genome PA0223 and PA4188. The active site residues in <i>Pa</i>DapA (PA1010) are indicated with yellow or purple colour as in C.</p