Paraoxonase 2 is down-regulated by the Pseudomonas aeruginosa quorumsensing signal N-(3-oxododecanoyl)-L-homoserine lactone and attenuates oxidative stress induced by pyocyanin.

Two virulence factors produced by Pseudomonas aeruginosa are pyocyanin and N-(3-oxododecanoyl)-L-homoserine lactone (3OC12). Pyocyanin damages host cells by generating ROS (reactive oxygen species). 3OC12 is a quorum-sensing signalling molecule which regulates bacterial gene expression and modulates host immune responses. PON2 (paraoxonase-2) is an esterase that inactivates 3OC12 and potentially attenuates Ps. aeruginosa virulence. Because increased intracellular Ca2+ initiates the degradation of PON2 mRNA and protein and 3OC12 causes increases in cytosolic Ca2+, we hypothesized that 3OC12 would also down-regulate PON2. 3OC12 and the Ca2+ ionophore A23187 caused a rapid cytosolic Ca2+ influx and down-regulated PON2 mRNA, protein and hydrolytic activity in A549 and EA.hy 926 cells. The decrease in PON2 hydrolytic activity was much more extensive and rapid than decreases in protein, suggesting a rapid post-translational mechanism which blocks PON2's hydrolytic activity. The Ca2+ chelator BAPTA/AM [1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid tetrakis(acetoxymethyl ester)] diminished the ability of 3OC12 to decrease PON2, demonstrating that the effects are mediated by Ca2+. PON2 also has antioxidative properties and we show that it protects cells from pyocyanin-induced oxidative stress. Knockdown of PON2 by transfecting cells with siRNA (small interfering RNA) rendered them more sensitive to, whereas overexpression of PON2 protected cells from, pyocyanin-induced ROS formation. Additionally, 3OC12 potentiated pyocyanin-induced ROS formation, presumably by inactivating PON2. These findings support a key role for PON2 in the defence against Ps. aeruginosa virulence, but also reveal a mechanism by which the bacterium may subvert the protection afforded by PON2.

The Basic Helix-Loop-Helix Gene Nato3 Drives Expression of Dopaminergic Neuron Transcription Factors in Neural Progenitors.

The floor plate of the developing midbrain gives rise to dopaminergic (DA) neurons, an important class of cells involved in Parkinson's disease (PD). Neural progenitors of the midbrain floor plate utilize key genes in transcriptional networks to drive dopamine neurogenesis. Identifying factors that promote dopaminergic neuron transcriptional networks can provide insight into strategies for therapies in PD. Using the chick embryo, we developed a quantitative PCR (qPCR) based method to assess the potential of a candidate factor to drive DA neuron gene expression, including the basic helix-loop-helix transcription factor Nato3 (Ferd3l). We then showed that overexpression of Nato3 in the developing chick mesencephalon produces a regionally dependent increase in genes associated with the DA neurogenesis, (such as Foxa2, Lmx1b and Shh) as well as DA neuron genes Nurr1 (an immature DA neuron marker) and mRNA expression of tyrosine hydroxylase (TH, a mature DA neuron marker). Interestingly, our data also showed that Nato3 is a potent regulator of Lmx1b by its broad induction of Lmx1b expression in neural progenitors of multiple regions of the CNS, including the midbrain and spinal cord. These data introduce a new, in vivo approach to identifying a gene that can drive DA transcriptional networks and provide the new insight that Nato3 can drive expression of key DA neuron genes, including Lmx1b, in neural progenitors.

Dominant role of paraoxonases in inactivation of the Pseudomonas aeruginosa quorum-sensing signal N-(3-oxododecanoyl)-L-homoserine lactone.

The pathogenic bacterium Pseudomonas aeruginosa causes serious infections in immunocompromised patients. N-(3-oxododecanoyl)-L-homoserine lactone (3OC12-HSL) is a key component of P. aeruginosa's quorum-sensing system and regulates the expression of many virulence factors. 3OC12-HSL was previously shown to be hydrolytically inactivated by the paraoxonase (PON) family of calcium-dependent esterases, consisting of PON1, PON2, and PON3. Here we determined the specific activities of purified human PONs for 3OC12-HSL hydrolysis, including the common PON1 polymorphic forms, and found they were in the following order: PON2 >> PON1(192R) > PON1(192Q) > PON3. PON2 exhibited a high specific activity of 7.6 +/- 0.4 micromols/min/mg at 10 microM 3OC12-HSL, making it the best PON2 substrate identified to date. By use of class-specific inhibitors, approximately 85 and 95% of the 3OC12-HSL lactonase activity were attributable to PON1 in mouse and human sera, respectively. In mouse liver homogenates, the activity was metal dependent, with magnesium- and manganese-dependent lactonase activities comprising 10 to 15% of the calcium-dependent activity. In mouse lung homogenates, all of the activity was calcium dependent. The calcium-dependent activities were irreversibly inhibited by extended EDTA treatment, implicating PONs as the major enzymes inactivating 3OC12-HSL. In human HepG2 and EA.hy 926 cell lysates, the 3OC12-HSL lactonase activity closely paralleled the PON2 protein levels after PON2 knockdown by small interfering RNA treatment of the cells. These findings suggest that PONs, particularly PON2, could be an important mechanism by which 3OC12-HSL is inactivated in mammals.

A single mutation in P450BM-3 enhances acyl homoserine lactone: acyl homoserine substrate binding selectivity nearly 250-fold.

Quorum sensing is the process by which bacteria alter gene regulation in response to their population density. The enzymatic inactivation of quorum signals has shown promise for use in genetically modified organisms resistant to pathogens. We recently characterized the ability of a cytochrome P450, P450BM-3, to oxidize the quorum sensing signals known as acyl homoserine lactones. The oxidation of the acyl homoserine lactones reduced their activity as quorum signals. The enzyme also oxidized the inactive lactonolysis products, acyl homoserines. The enzyme showed similar binding affinity for the acyl homoserine lactones and acyl homoserines. The latter reaction may lead to problems when lactonases and the P450-dependent system are used in tandem, as oxidation of the acyl homoserines produced by lactonolysis in vivo may compete with acyl homoserine lactone oxidation by the cytochrome P450. We report here that a single mutation (R47S) in P450BM-3 is capable of increasing the acyl homoserine lactone: acyl homoserine substrate binding selectivity of the enzyme nearly 250-fold, reducing the potential for competition by acyl homoserines and significantly enhancing the potential for use of P450BM-3 as part of a pathogen resistance system in genetically modified crops.

Cloning, expression and characterization of a fast self-sufficient P450: CYP102A5 from Bacillus cereus.

CYP102s represent a family of natural self-sufficient fusions of cytochrome P450 and cytochrome P450 reductase found in some bacteria. One member of this family, named CYP102A1 or more traditionally P450BM-3, has been widely studied as a model of human P450 cytochromes. Remarkable detail of P450 structure and function has been revealed using this highly efficient enzyme. The recent rapid expansion of microbial genome sequences has revealed many relatives of CYP102A1, but to date only two from Bacillus subtilis have been characterized. We report here the cloning and expression of CYP102A5, a new member of this family that is very closely related to CYP102A4 from Bacillus anthracis. Characterization of the substrate specificity of CYP102A5 shows that it, like the other CYP102s, will metabolize saturated and unsaturated fatty acids as well as N-acylamino acids. CYP102A5 catalyzes very fast substrate oxidation, showing one of the highest turnover rates for any P450 monooxygenase studied so far. It does so with more specificity than other CYP102s, yielding primarily omega-1 and omega-2 hydroxylated products. Measurement of the rate of electron transfer through the reductase domain reveals that it is significantly faster in CYP102A5 than in CYP102A1, providing a likely explanation for the increased monooxygenation rate. The availability of this new, very fast fusion P450 will provide a great tool for comparative structure-function studies between CYP102A5 and the other characterized CYP102s.

Bacillus megaterium CYP102A1 oxidation of acyl homoserine lactones and acyl homoserines.

Quorum sensing, the ability of bacteria to sense their own population density through the synthesis and detection of small molecule signals, has received a great deal of attention in recent years. Acyl homoserine lactones (AHLs) are a major class of quorum sensing signaling molecules. In nature, some bacteria that do not synthesize AHLs themselves have developed the ability to degrade these compounds by cleaving the amide bond or the lactone ring. By inactivating this signal used by competing bacteria, the degrading microbe is believed to gain a competitive advantage. In this work we report that CYP102A1, a widely studied cytochrome P450 from Bacillus megaterium, is capable of very efficient oxidation of AHLs and their lactonolysis products acyl homoserines. The previously known substrates for this enzyme, fatty acids, can also be formed in nature by hydrolysis of the amide of AHLs, so CYP102A1 is capable of inactivating the active parent compound and the products of both known pathways for AHL inactivation observed in nature. AHL oxidation primarily takes place at the omega-1, omega-2, and omega-3 carbons of the acyl chain, similar to this enzyme's well-known activity on fatty acids. Acyl homoserines and their lactones are better substrates for CYP102A1 than fatty acids. Bioassay of the quorum sensing activity of oxidation products reveals that the subterminally hydroxylated AHLs exhibit quorum sensing activity, but are 18-fold less active than the parent compound. In vivo, B. megaterium inactivates AHLs by a CYP102A1 dependent mechanism that must involve additional components that further sequester or metabolize the products, eliminating their quorum sensing activity. Cytochrome P450 oxidation of AHLs represents an important new mechanism of quorum quenching.

Interactions of substrates at the surface of P450s can greatly enhance substrate potency.

Cytochrome P450s are a superfamily of heme containing enzymes that use molecular oxygen and electrons from reduced nicotinamide cofactors to monooxygenate organic substrates. The fatty acid hydroxylase P450BM-3 has been particularly widely studied due to its stability, high activity, similarity to mammalian P450s, and presence of a cytochrome P450 reductase domain that allows the enzyme to directly receive electrons from NADPH without a requirement for additional redox proteins. We previously characterized the substrate N-palmitoylglycine, which found extensive use in studies of P450BM-3 due to its high affinity, high turnover number, and increased solubility as compared to fatty acid substrates. Here, we report that even higher affinity substrates can be designed by acylation of other amino acids, resulting in P450BM-3 substrates with dissociation constants below 100 nM. N-Palmitoyl-l-leucine and N-palmitoyl-l-methionine were found to have the highest affinity, with dissociation constants of less than 8 nM and turnover numbers similar to palmitic acid and N-palmitoylglycine. The interactions of the amino acid side chains with a hydrophobic pocket near R47, as revealed by our crystal structure determination of N-palmitoyl-l-methionine bound to the heme domain of P450BM-3, appears to be responsible for increasing the affinity of substrates. The side chain of R47, previously shown to be important in interactions with negatively charged substrates, does not interact strongly with N-palmitoyl-l-methionine and is found positioned at the enzyme-solvent interface. These are the tightest binding substrates for P450BM-3 reported to date, and the affinity likely approaches the maximum attainable affinity for the binding of substrates of this size to P450BM-3.

L-Canavanine made by Medicago sativa interferes with quorum sensing in Sinorhizobium meliloti.

Sinorhizobium meliloti is a gram-negative soil bacterium, capable of establishing a nitrogen-fixing symbiosis with its legume host, alfalfa (Medicago sativa). Quorum sensing plays a crucial role in this symbiosis, where it influences the nodulation process and the synthesis of the symbiotically important exopolysaccharide II (EPS II). S. meliloti has three quorum-sensing systems (Sin, Tra, and Mel) that use N-acyl homoserine lactones as their quorum-sensing signal molecule. Increasing evidence indicates that certain eukaryotic hosts involved in symbiotic or pathogenic relationships with gram-negative bacteria produce quorum-sensing-interfering (QSI) compounds that can cross-communicate with the bacterial quorum-sensing system. Our studies of alfalfa seed exudates suggested the presence of multiple signal molecules capable of interfering with quorum-sensing-regulated gene expression in different bacterial strains. In this work, we choose one of these QSI molecules (SWI) for further characterization. SWI inhibited violacein production, a phenotype that is regulated by quorum sensing in Chromobacterium violaceum. In addition, this signal molecule also inhibits the expression of the S. meliloti exp genes, responsible for the production of EPS II, a quorum-sensing-regulated phenotype. We identified this molecule as l-canavanine, an arginine analog, produced in large quantities by alfalfa and other legumes.