Everett C. Pesci

Functions Required for Extracellular Quinolone Signaling by Pseudomonas aeruginosa

A set of 30 mutants exhibiting reduced production of the phenazine poison pyocyanin were isolated following transposon mutagenesis of Pseudomonas aeruginosa PAO1. The mutants could be subdivided into those with defects in the primary phenazine biosynthetic pathway and those with more pleiotropic defects. The largest set of pleiotropic mutations blocked the production of the extracellular Pseudomonas quinolone signal (PQS), a molecule required for the synthesis of secondary metabolites and extracellular enzymes. Most of these pqs mutations affected genes which appear to encode PQS biosynthetic functions, although a transcriptional regulator and an apparent response effector were also represented. Two of the genes required for PQS synthesis (phnA and phnB) had previously been assumed to encode phenazine biosynthetic functions. The transcription of one of the genes required for PQS synthesis (PA2587/pqsH) was regulated by the LasI/R quorum-sensing system, thereby linking quorum sensing and PQS regulation. Others of the pleiotropic phenazine-minus mutations appear to inactivate novel components of the quorum-sensing regulatory network, including one regulator (np20) previously shown to be required for virulence in neutropenic mice.

Autolysis and Autoaggregation in Pseudomonas aeruginosa Colony Morphology Mutants

Two distinctive colony morphologies were noted in a collection of Pseudomonas aeruginosa transposon insertion mutants. One set of mutants formed wrinkled colonies of autoaggregating cells. Suppressor analysis of a subset of these mutants showed that this was due to the action of the regulator WspR and linked this regulator (and the chemosensory pathway to which it belongs) to genes that encode a putative fimbrial adhesin required for biofilm formation. WspR homologs, related in part by a shared GGDEF domain, regulate cell surface factors, including aggregative fimbriae and exopolysaccharides, in diverse bacteria. The second set of distinctive insertion mutants formed colonies that lysed at their center. Strains with the most pronounced lysis overproduced the Pseudomonas quinolone signal (PQS), an extracellular signal that interacts with quorum sensing. Autolysis was suppressed by mutation of genes required for PQS biosynthesis, and in one suppressed mutant, autolysis was restored by addition of synthetic PQS. The mechanism of autolysis may involve activation of the endogenous prophage and phage-related pyocins in the genome of strain PAO1. The fact that PQS levels correlated with autolysis suggests a fine balance in natural populations of P. aeruginosa between survival of the many and persistence of the few.

Regulation of Pseudomonas Quinolone Signal Synthesis in Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen that causes chronic lung infections in cystic fibrosis patients and is a major source of nosocomial infections. This bacterium controls many virulence factors by using two quorum-sensing systems, las and rhl. The las system is composed of the LasR regulator protein and its cell-to-cell signal, N-(3-oxododecanoyl) homoserine lactone, and the rhl system is composed of RhlR and the signal N-butyryl homoserine lactone. A third intercellular signal, the Pseudomonas quinolone signal (PQS; 2-heptyl-3-hydroxy-4-quinolone), also regulates numerous virulence factors. PQS synthesis requires the expression of multiple operons, one of which is pqsABCDE. Previous experiments showed that the transcription of this operon, and therefore PQS production, is negatively regulated by the rhl quorum-sensing system and positively regulated by the las quorum-sensing system and PqsR (also known as MvfR), a LysR-type transcriptional regulator protein. With the use of DNA mobility shift assays and beta-galactosidase reporter fusions, we have studied the regulation of pqsR and its relationship to pqsA, lasR, and rhlR. We show that PqsR binds the promoter of pqsA and that this binding increases dramatically in the presence of PQS, implying that PQS acts as a coinducer for PqsR. We have also mapped the transcriptional start site for pqsR and found that the transcription of pqsR is positively regulated by lasR and negatively regulated by rhlR. These results suggest that a regulatory chain occurs where pqsR is under the control of LasR and RhlR and where PqsR in turn controls pqsABCDE, which is required for the production of PQS.

Farnesol, a common sesquiterpene, inhibits PQS production in Pseudomonas aeruginosa.

Farnesol is a sesquiterpene produced by many organisms, including the fungus Candida albicans. Here, we report that the addition of farnesol to cultures of Pseudomonas aeruginosa, an opportunistic human bacterial pathogen, leads to decreased production of the Pseudomonas quinolone signal (PQS) and the PQS‐controlled virulence factor, pyocyanin. Within 15 min of farnesol addition, decreased transcript levels of pqsA, the first gene in the PQS biosynthetic operon, were observed. Transcript levels of pqsR (mvfR), which encodes the transcription factor that positively regulates pqsA, were unaffected. An Escherichia coli strain producing PqsR and containing the pqsA promoter fused to lacZ similarly showed that farnesol inhibited PQS‐stimulated transcription. Electrophoretic mobility shift assays showed that, like PQS, farnesol stimulated PqsR binding to the pqsA promoter at a previously characterized LysR binding site, suggesting that farnesol promoted a non‐productive interaction between PqsR and the pqsA promoter. Growth with C. albicans leads to decreased production of PQS and pyocyanin by P. aeruginosa, suggesting that the amount of farnesol produced by the fungus is sufficient to impact P. aeruginosa PQS signalling. Related isoprenoid compounds, but not other long‐chain alcohols, also inhibited PQS production at micromolar concen‐trations, suggesting that related compounds may participate in interspecies interactions with P. aeruginosa.

Interference with Pseudomonas quinolone signal synthesis inhibits virulence factor expression by Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen that controls numerous virulence factors through intercellular signals. This bacterium has two quorum-sensing systems (las and rhl), which act through the intercellular signals N-(3-oxododecanoyl)-l-homoserine lactone (3-oxo-C12-HSL) and N-butyryl-l-homoserine lactone (C4-HSL), respectively. P. aeruginosa also produces a third intercellular signal that is involved in virulence factor regulation. This signal, 2-heptyl-3-hydroxy-4-quinolone [referred to as the Pseudomonas quinolone signal (PQS)], is a secondary metabolite that is part of the P. aeruginosa quorum-sensing hierarchy. PQS can induce both lasB (encodes LasB elastase) and rhlI (encodes the C4-HSL synthase) in P. aeruginosa and is produced maximally during the late stationary phase of growth. Because PQS is an intercellular signal that is part of the quorum-sensing hierarchy and controls multiple virulence factors, we began basic studies designed to elucidate its biosynthetic pathway. First, we present data that strongly suggest that anthranilate is a precursor for PQS. P. aeruginosa converted radiolabeled anthranilate into radioactive PQS, which was bioactive. We also found that an anthranilate analog (methyl anthranilate) would inhibit the production of PQS. This analog was then shown to have a major negative effect on elastase production by P. aeruginosa. These data provide evidence that precursors of intercellular signals may provide viable targets for the development of therapeutic treatments that will reduce P. aeruginosa virulence.

Dueling quorum sensing systems in Pseudomonas aeruginosa control the production of the Pseudomonas quinolone signal (PQS)

The opportunistic human pathogen Pseudomonas aeruginosa regulates the production of numerous virulence factors via the action of two separate but coordinated quorum sensing systems, las and rhl. These systems control the transcription of genes in response to population density through the intercellular signals N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C(12)-HSL) and N-(butanoyl)-L-homoserine lactone (C(4)-HSL). A third P. aeruginosa signal, 2-heptyl-3-hydroxy-4-quinolone [Pseudomonas quinolone signal (PQS)], also plays a significant role in the transcription of multiple P. aeruginosa virulence genes. PQS is intertwined in the P. aeruginosa quorum sensing hierarchy with its production and bioactivity requiring the las and rhl quorum sensing systems, respectively. This report presents a preliminary transcriptional analysis of pqsA, the first gene of the recently discovered PQS biosynthetic gene cluster. We show that pqsA transcription required pqsR, a transcriptional activator protein encoded within the PQS biosynthetic gene cluster. It was also found that the transcription of pqsA and subsequent production of PQS was induced by the las quorum sensing system and repressed by the rhl quorum sensing system. In addition, PQS production was dependent on the ratio of 3-oxo-C(12)-HSL to C(4)-HSL, suggesting a regulatory balance between quorum sensing systems. These data are an important early step toward understanding the regulation of PQS synthesis and the role of PQS in P. aeruginosa intercellular signaling.

The Influence of Iron on Pseudomonas aeruginosa Physiology A REGULATORY LINK BETWEEN IRON AND QUORUM SENSING

In iron-replete environments, the Pseudomonas aeruginosa Fur (ferric uptake regulator) protein represses expression of two small regulatory RNAs encoded by prrF1 and prrF2. Here we describe the effects of iron and PrrF regulation on P. aeruginosa physiology. We show that PrrF represses genes encoding enzymes for the degradation of anthranilate (i.e. antABC), a precursor of the Pseudomonas quinolone signal (PQS). Under iron-limiting conditions, PQS production was greatly decreased in a ΔprrF1,2 mutant as compared with wild type. The addition of anthranilate to the growth medium restored PQS production to the ΔprrF1,2 mutant, indicating that its defect in PQS production is a consequence of anthranilate degradation. PA2511 was shown to encode an anthranilate-dependent activator of the ant genes and was subsequently renamed antR. AntR was not required for regulation of antA by PrrF but was required for optimal iron activation of antA. Furthermore, iron was capable of activating both antA and antR in a ΔprrF1,2 mutant, indicating the presence of two distinct yet overlapping pathways for iron activation of antA (AntR-dependent and PrrF-dependent). Additionally, several quorum-sensing regulators, including PqsR, influenced antA expression, demonstrating that regulation of anthranilate metabolism is intimately woven into the quorum-sensing network of P. aeruginosa. Overall, our data illustrate the extensive control that both iron regulation and quorum sensing exercise in basic cellular physiology, underlining how intermediary metabolism can affect the regulation of virulence factors in P. aeruginosa.

Two Distinct Pathways Supply Anthranilate as a Precursor of the Pseudomonas Quinolone Signal

Pseudomonas aeruginosa is an opportunistic pathogen that causes serious infections in immunocompromised patients and those with cystic fibrosis (CF). This gram-negative bacterium uses multiple cell-to-cell signals to control numerous cellular functions and virulence. One of these signals is 2-heptyl-3-hydroxy-4-quinolone, which is referred to as the Pseudomonas quinolone signal (PQS). This signal functions as a coinducer for a transcriptional regulator (PqsR) to positively control multiple virulence genes and its own synthesis. PQS production is required for virulence in multiple models of infection, and it has been shown to be produced in the lungs of CF patients infected by P. aeruginosa. One of the precursor compounds from which PQS is synthesized is the metabolite anthranilate. This compound can be derived from the conversion of chorismate to anthranilate by an anthranilate synthase or through the degradation of tryptophan via the anthranilate branch of the kynurenine pathway. In this study, we present data which help to define the kynurenine pathway in P. aeruginosa and show that the kynurenine pathway serves as a critical source of anthranilate for PQS synthesis. We also show that the kyn pathway genes are induced during growth with tryptophan and that they are autoregulated by kynurenine. This study provides solid foundations for the understanding of how P. aeruginosa produces the anthranilate that serves as a precursor to PQS and other 4-quinolones.

Pseudomonas aeruginosa relA contributes to virulence in Drosophila melanogaster.

ABSTRACT The stringent response is a mechanism by which bacteria adapt to nutritional deficiencies through the production of the guanine nucleotides ppGpp and pppGpp, produced by the RelA enzyme. We investigated the role of the relA gene in the ability of an extracellular pathogen, Pseudomonas aeruginosa, to cause infection. Strains lacking the relA gene were created from the prototypical laboratory strain PAO1 as well as the mucoid cystic fibrosis isolate 6106, which lacks functional quorum-sensing systems. The absence of relA abolished the production of ppGpp and pppGpp under conditions of amino acid starvation. We found that strains lacking relA exhibited reduced virulence in a D. melanogaster feeding assay. In conditions of low magnesium, the relA gene enhanced production of the cell-cell signal N-[3-oxododecanoyl]-l-homoserine lactone, whereas relA reduced the production of the 2-heptyl-3-hydroxy-4-quinolone signal during serine hydroxamate induction of the stringent response. In the relA mutant, alterations in the Pseudomonas quinolone system pathways seemed to increase the production of pyocyanin and decrease the production of elastase. Deletion of relA also resulted in reduced levels of the RpoS sigma factor. These results suggest that adjustment of cellular ppGpp and pppGpp levels could be an important regulatory mechanism in P. aeruginosa adaptation in pathogenic relationships.

PqsE functions independently of PqsR-Pseudomonas quinolone signal and enhances the rhl quorum-sensing system.

Pseudomonas aeruginosa is an opportunistic pathogen that causes both acute and chronic infections in immunocompromised individuals. This gram-negative bacterium produces a battery of virulence factors that allow it to infect and survive in many different hostile environments. The control of many of these virulence factors falls under the influence of one of three P. aeruginosa cell-to-cell signaling systems. The focus of this study, the quinolone signaling system, functions through the Pseudomonas quinolone signal (PQS), previously identified as 2-heptyl-3-hydroxy-4-quinolone. This signal binds to and activates the LysR-type transcriptional regulator PqsR (also known as MvfR), which in turn induces the expression of the pqsABCDE operon. The first four genes of this operon are required for PQS synthesis, but the fifth gene, pqsE, is not. The function of the pqsE gene is not known, but it is required for the production of multiple PQS-controlled virulence factors and for virulence in multiple models of infection. In this report, we show that PqsE can activate PQS-controlled genes in the absence of PqsR and PQS. Our data also suggest that the regulatory activity of PqsE requires RhlR and indicate that a pqsE mutant can be complemented for pyocyanin production by a large excess of exogenous N-butyryl homoserine lactone (C4-HSL). Finally, we show that PqsE enhances the ability of Escherichia coli expressing RhlR to respond to C4-HSL. Overall, our data lead us to conclude that PqsE functions as a regulator that is independent of PqsR and PQS but dependent on the rhl quorum-sensing system.