The Quorum-Sensing Molecules Farnesol/Homoserine Lactone and Dodecanol Operate via Distinct Modes of Action in Candida albicans.

Living as a commensal, Candida albicans must adapt and respond to environmental cues generated by the mammalian host and by microbes comprising the natural flora. These signals have opposing effects on C. albicans, with host cues promoting the yeast-to-hyphal transition and bacteria-derived quorum-sensing molecules inhibiting hyphal development. Hyphal development is regulated through modulation of the cyclic AMP (cAMP)/protein kinase A (PKA) signaling pathway, and it has been postulated that quorum-sensing molecules can affect filamentation by inhibiting the cAMP pathway. Here, we show that both farnesol and 3-oxo-C(12)-homoserine lactone, a quorum-sensing molecule secreted by Pseudomonas aeruginosa, block hyphal development by affecting cAMP signaling; they both directly inhibited the activity of the Candida adenylyl cyclase, Cyr1p. In contrast, the 12-carbon alcohol dodecanol appeared to modulate hyphal development and the cAMP signaling pathway without directly affecting the activity of Cyr1p. Instead, we show that dodecanol exerted its effects through a mechanism involving the C. albicans hyphal repressor, Sfl1p. Deletion of SFL1 did not affect the response to farnesol but did interfere with the response to dodecanol. Therefore, quorum sensing in C. albicans is mediated via multiple mechanisms of action. Interestingly, our experiments raise the possibility that the Burkholderia cenocepacia diffusible signal factor, BDSF, also mediates its effects via Sfl1p, suggesting that dodecanol's mode of action, but not farnesol or 3-oxo-C(12)-homoserine lactone, may be used by other quorum-sensing molecules.

Degradation of pathogen quorum-sensing molecules by soil bacteria: a preventive and curative biological control mechanism.

Abstract The plasmid pME6863, carrying the aiiA gene from the soil bacterium Bacillus sp. A24 that encodes a lactonase enzyme able to degrade N-acyl-homoserine lactones (AHLs), was introduced into the rhizosphere isolate Pseudomonas fluorescens P3. This strain is not an effective biological control agent against plant pathogens. The transformant P. fluorescens P3/pME6863 acquired the ability to degrade AHLs. In planta, P. fluorescens P3/pME6863 significantly reduced potato soft rot caused by Erwinia carotovora and crown gall of tomato caused by Agrobacterium tumefaciens to a similar level as Bacillus sp. A24. Little or no disease reduction was observed for the wild-type strain P3 carrying the vector plasmid without aiiA. Suppression of potato soft rot was observed even when the AHL-degrading P. fluorescens P3/pME6863 was applied to tubers 2 days after the pathogen, indicating that biocontrol was not only preventive but also curative. When antagonists were applied individually with the bacterial plant pathogens, biocontrol activity of the AHL degraders was greater than that observed with several Pseudomonas 2,4-diacetylphloroglucinol-producing strains and with Pseudomonas chlororaphis PCL1391, which relies on production of phenazine antibiotic for disease suppression. Phenazine production by this well characterized biological control strain P. chlororaphis PCL1391 is regulated by AHL-mediated quorum sensing. When P. chlororaphis PCL1391 was co-inoculated with P. fluorescens P3/pME6863 in a strain mixture, the AHL degrader interfered with the normally excellent ability of the antibiotic producer to suppress tomato vascular wilt caused by Fusarium oxysporum f. sp. lycopersici. Our results demonstrate AHL degradation as a novel biocontrol mechanism, but also demonstrate the potential for non-target interactions that can interfere with the biocontrol efficacy of other strains.

A quorum sensing regulated small volatile molecule reduces acute virulence and promotes chronic infection phenotypes.

A significant number of environmental microorganisms can cause serious, even fatal, acute and chronic infections in humans. The severity and outcome of each type of infection depends on the expression of specific bacterial phenotypes controlled by complex regulatory networks that sense and respond to the host environment. Although bacterial signals that contribute to a successful acute infection have been identified in a number of pathogens, the signals that mediate the onset and establishment of chronic infections have yet to be discovered. We identified a volatile, low molecular weight molecule, 2-amino acetophenone (2-AA), produced by the opportunistic human pathogen Pseudomonas aeruginosa that reduces bacterial virulence in vivo in flies and in an acute mouse infection model. 2-AA modulates the activity of the virulence regulator MvfR (multiple virulence factor regulator) via a negative feedback loop and it promotes the emergence of P. aeruginosa phenotypes that likely promote chronic lung infections, including accumulation of lasR mutants, long-term survival at stationary phase, and persistence in a Drosophila infection model. We report for the first time the existence of a quorum sensing (QS) regulated volatile molecule that induces bistability phenotype by stochastically silencing acute virulence functions in P. aeruginosa. We propose that 2-AA mediates changes in a subpopulation of cells that facilitate the exploitation of dynamic host environments and promote gene expression changes that favor chronic infections.

Quorum-sensing systems in staphylococci as therapeutic targets.

The staphylococci are an ever-present threat in our world, capable of causing a wide range of infections, and are a persistent presence in the clinical environment. As the number of antimicrobial compounds effective against staphylococci decreases, because of the acquisition and spread of antibiotic resistance, there is a growing need for novel therapeutic molecules. Intra and inter-species communication (quorum sensing) is a biologically significant phenomenon that has been associated with virulence, intracellular survival, and biofilm formation. Quorum sensing molecules of staphylococci and other species (e.g. Pseudomonas aeruginosa) can inhibit virulence factor production and/or growth of staphylococci, leading to the possibility that interference with staphylococcal quorum-sensing systems could be a way of controlling the diverse infections caused by the staphylococci. In this article, we discuss the potential of quorum-sensing systems of staphylococci as therapeutic targets.

Transcriptional control of the hydrogen cyanide biosynthetic genes hcnABC by the anaerobic regulator ANR and the quorum-sensing regulators LasR and RhlR in Pseudomonas aeruginosa.

Virulence factors of Pseudomonas aeruginosa include hydrogen cyanide (HCN). This secondary metabolite is maximally produced at low oxygen tension and high cell densities during the transition from exponential to stationary growth phase. The hcnABC genes encoding HCN synthase were identified on a genomic fragment complementing an HCN-deficient mutant of P. aeruginosa PAO1. The hcnA promoter was found to be controlled by the FNR-like anaerobic regulator ANR and by the quorum-sensing regulators LasR and RhlR. Primer extension analysis revealed two transcription starts, T1 and T2, separated by 29 bp. Their function was confirmed by transcriptional lacZ fusions. The promoter sequence displayed an FNR/ANR box at -42.5 bp upstream of T2 and a lux box centered around -42.5 bp upstream of T1. Expression of the hcn genes was completely abolished when this lux box was deleted or inactivated by two point mutations in conserved nucleotides. The lux box was recognized by both LasR [activated by N-(oxododecanoyl)-homoserine lactone] and RhlR (activated by N-butanoyl-homoserine lactone), as shown by expression experiments performed in quorum-sensing-defective P. aeruginosa mutants and in the N-acyl-homoserine lactone-negative heterologous host P. fluorescens CHA0. A second, less conserved lux box lying 160 bp upstream of T1 seems to account for enhanced quorum-sensing-dependent expression. Without LasR and RhlR, ANR could not activate the hcn promoter. Together, these data indicate that expression of the hcn promoter from T1 can occur under quorum-sensing control alone. Enhanced expression from T2 appears to rely on a synergistic action between LasR, RhlR, and ANR.

A quorum sensing small volatile molecule promotes antibiotic tolerance in bacteria.

Bacteria can be refractory to antibiotics due to a sub-population of dormant cells, called persisters that are highly tolerant to antibiotic exposure. The low frequency and transience of the antibiotic tolerant "persister" trait has complicated elucidation of the mechanism that controls antibiotic tolerance. In this study, we show that 2' Amino-acetophenone (2-AA), a poorly studied but diagnostically important small, volatile molecule produced by the recalcitrant gram-negative human pathogen Pseudomonas aeruginosa, promotes antibiotic tolerance in response to quorum-sensing (QS) signaling. Our results show that 2-AA mediated persister cell accumulation occurs via alteration of the expression of genes involved in the translational capacity of the cell, including almost all ribosomal protein genes and other translation-related factors. That 2-AA promotes persisters formation also in other emerging multi-drug resistant pathogens, including the non 2-AA producer Acinetobacter baumannii implies that 2-AA may play an important role in the ability of gram-negative bacteria to tolerate antibiotic treatments in polymicrobial infections. Given that the synthesis, excretion and uptake of QS small molecules is a common hallmark of prokaryotes, together with the fact that the translational machinery is highly conserved, we posit that modulation of the translational capacity of the cell via QS molecules, may be a general, widely distributed mechanism that promotes antibiotic tolerance among prokaryotes.

Negative control of quorum sensing by RpoN (sigma54) in Pseudomonas aeruginosa PAO1.

In Pseudomonas aeruginosa PAO1, the expression of several virulence factors such as elastase, rhamnolipids, and hydrogen cyanide depends on quorum-sensing regulation, which involves the lasRI and rhlRI systems controlled by N-(3-oxododecanoyl)-L-homoserine lactone and N-butyryl-L-homoserine lactone, respectively, as signal molecules. In rpoN mutants lacking the transcription factor sigma(54), the expression of the lasR and lasI genes was elevated at low cell densities, whereas expression of the rhlR and rhlI genes was markedly enhanced throughout growth by comparison with the wild type and the complemented mutant strains. As a consequence, the rpoN mutants had elevated levels of both signal molecules and overexpressed the biosynthetic genes for elastase, rhamnolipids, and hydrogen cyanide. The quorum-sensing regulatory protein QscR was not involved in the negative control exerted by RpoN. By contrast, in an rpoN mutant, the expression of the gacA global regulatory gene was significantly increased during the entire growth cycle, whereas another global regulatory gene, vfr, was downregulated at high cell densities. In conclusion, it appears that GacA levels play an important role, probably indirectly, in the RpoN-dependent modulation of the quorum-sensing machinery of P. aeruginosa.

Impact of quorum sensing on fitness of Pseudomonas aeruginosa.

In Pseudomonas aeruginosa, cell-cell communication based on N-acyl-homoserine lactone (AHL) signal molecules (termed quorum sensing) is known to control the production of extracellular virulence factors. Hence, in pathogenic interactions with host organisms, the quorum-sensing (QS) machinery can confer a selective advantage on P. aeruginosa. However, as shown by transcriptomic and proteomic studies, many intracellular metabolic functions are also regulated by quorum sensing. Some of these serve to regenerate the AHL precursors methionine and S-adenosyl-methionine and to degrade adenosine via inosine and hypoxanthine. The fact that a significant percentage of clinical and environmental isolates of P. aeruginosa is defective for QS because of mutation in the major QS regulatory gene lasR, raises the question of whether the QS machinery can have a negative impact on the organism's fitness. In vitro, lasR mutants have a higher probability to escape lytic death in stationary phase under alkaline conditions than has the QS-proficient wild type. Similar selective forces might also operate in natural environments.

Two GacA-dependent small RNAs modulate the quorum-sensing response in Pseudomonas aeruginosa.

In Pseudomonas aeruginosa, the GacS/GacA two-component system positively controls the quorum-sensing machinery and the expression of extracellular products via two small regulatory RNAs, RsmY and RsmZ. An rsmY rsmZ double mutant and a gacA mutant were similarly impaired in the synthesis of the quorum-sensing signal N-butanoyl-homoserine lactone, the disulfide bond-forming enzyme DsbA, and the exoproducts hydrogen cyanide, pyocyanin, elastase, chitinase (ChiC), and chitin-binding protein (CbpD). Both mutants showed increased swarming ability, azurin release, and early biofilm development.

Quorum-sensing-negative (lasR) mutants of Pseudomonas aeruginosa avoid cell lysis and death.

In Pseudomonas aeruginosa, N-acylhomoserine lactone signals regulate the expression of several hundreds of genes, via the transcriptional regulator LasR and, in part, also via the subordinate regulator RhlR. This regulatory network termed quorum sensing contributes to the virulence of P. aeruginosa as a pathogen. The fact that two supposed PAO1 wild-type strains from strain collections were found to be defective for LasR function because of independent point mutations in the lasR gene led to the hypothesis that loss of quorum sensing might confer a selective advantage on P. aeruginosa under certain environmental conditions. A convenient plate assay for LasR function was devised, based on the observation that lasR mutants did not grow on adenosine as the sole carbon source because a key degradative enzyme, nucleoside hydrolase (Nuh), is positively controlled by LasR. The wild-type PAO1 and lasR mutants showed similar growth rates when incubated in nutrient yeast broth at pH 6.8 and 37 degrees C with good aeration. However, after termination of growth during 30 to 54 h of incubation, when the pH rose to > or = 9, the lasR mutants were significantly more resistant to cell lysis and death than was the wild type. As a consequence, the lasR mutant-to-wild-type ratio increased about 10-fold in mixed cultures incubated for 54 h. In a PAO1 culture, five consecutive cycles of 48 h of incubation sufficed to enrich for about 10% of spontaneous mutants with a Nuh(-) phenotype, and five of these mutants, which were functionally complemented by lasR(+), had mutations in lasR. The observation that, in buffered nutrient yeast broth, the wild type and lasR mutants exhibited similar low tendencies to undergo cell lysis and death suggests that alkaline stress may be a critical factor providing a selective survival advantage to lasR mutants.

Auto Poisoning of the Respiratory Chain by a Quorum-Sensing-Regulated Molecule Favors Biofilm Formation and Antibiotic Tolerance.

Bacterial programmed cell death and quorum sensing are direct examples of prokaryote group behaviors, wherein cells coordinate their actions to function cooperatively like one organism for the benefit of the whole culture. We demonstrate here that 2-n-heptyl-4-hydroxyquinoline-N-oxide (HQNO), a Pseudomonas aeruginosa quorum-sensing-regulated low-molecular-weight excreted molecule, triggers autolysis by self-perturbing the electron transfer reactions of the cytochrome bc1 complex. HQNO induces specific self-poisoning by disrupting the flow of electrons through the respiratory chain at the cytochrome bc1 complex, causing a leak of reducing equivalents to O2 whereby electrons that would normally be passed to cytochrome c are donated directly to O2. The subsequent mass production of reactive oxygen species (ROS) reduces membrane potential and disrupts membrane integrity, causing bacterial cell autolysis and DNA release. DNA subsequently promotes biofilm formation and increases antibiotic tolerance to beta-lactams, suggesting that HQNO-dependent cell autolysis is advantageous to the bacterial populations. These data identify both a new programmed cell death system and a novel role for HQNO as a critical inducer of biofilm formation and antibiotic tolerance. This newly identified pathway suggests intriguing mechanistic similarities with the initial mitochondrial-mediated steps of eukaryotic apoptosis.

Peroxisome proliferator-activated receptors mediate host cell proinflammatory responses to Pseudomonas aeruginosa autoinducer.

The pathogenic bacterium Pseudomonas aeruginosa utilizes the 3-oxododecanoyl homoserine lactone (3OC(12)-HSL) autoinducer as a signaling molecule to coordinate the expression of virulence genes through quorum sensing. 3OC(12)-HSL also affects responses in host cells, including the upregulation of genes encoding inflammatory cytokines. This proinflammatory response may exacerbate underlying disease during P. aeruginosa infections. The specific mechanism(s) through which 3OC(12)-HSL influences host responses is unclear, and no mammalian receptors for 3OC(12)-HSL have been identified to date. Here, we report that 3OC(12)-HSL increases mRNA levels for a common panel of proinflammatory genes in murine fibroblasts and human lung epithelial cells. To identify putative 3OC(12)-HSL receptors, we examined the expression patterns of a panel of nuclear hormone receptors in these two cell lines and determined that both peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) and PPARgamma were expressed. 3OC(12)-HSL functioned as an agonist of PPARbeta/delta transcriptional activity and an antagonist of PPARgamma transcriptional activity and inhibited the DNA binding ability of PPARgamma. The proinflammatory effect of 3OC(12)-HSL in lung epithelial cells was blocked by the PPARgamma agonist rosiglitazone, suggesting that 3OC(12)-HSL and rosiglitazone are mutually antagonistic negative and positive regulators of PPARgamma activity, respectively. These data identify PPARbeta/delta and PPARgamma as putative mammalian 3OC(12)-HSL receptors and suggest that PPARgamma agonists may be employed as anti-inflammatory therapeutics for P. aeruginosa infections.

Allorestricted T lymphocytes with a high avidity T-cell receptor towards NY-ESO-1 have potent anti-tumor activity.

The cancer-testis antigen NY-ESO-1 has been targeted as a tumor-associated antigen by immunotherapeutical strategies, such as cancer vaccines. The prerequisite for a T-cell-based therapy is the induction of T cells capable of recognizing the NY-ESO-1-expressing tumor cells. In this study, we generated human T lymphocytes directed against the immunodominant NY-ESO-1(157-165) epitope known to be naturally presented with HLA-A*0201. We succeeded to isolate autorestricted and allorestricted T lymphocytes with low, intermediate or high avidity TCRs against the NY-ESO-1 peptide. The avidity of the established CTL populations correlated with their capacity of lysing HLA-A2-positive, NY-ESO-1-expressing tumor cell lines derived from different origins, e.g. melanoma and myeloma. The allorestricted NY-ESO-1-specific T lymphocytes displayed TCRs with the highest avidity and best anti-tumor recognition activity. TCRs derived from allorestricted, NY-ESO-1-specific T cells may be useful reagents for redirecting primary T cells by TCR gene transfer and, therefore, may facilitate the development of adoptive transfer regimens based on TCR-transduced T cells for the treatment of NY-ESO-1-expressing hematological malignancies and solid tumors.

PEGylating a bacteriophage endolysin inhibits its bactericidal activity.

ABSTRACT: Bacteriophage endolysins (lysins) bind to a cell wall substrate and cleave peptidoglycan, resulting in hypotonic lysis of the phage-infected bacteria. When purified lysins are added externally to Gram-positive bacteria they mediate rapid death by the same mechanism. For this reason, novel therapeutic strategies have been developed using such enzybiotics. However, like other proteins introduced into mammalian organisms, they are quickly cleared from systemic circulation. PEGylation has been used successfully to increase the in vivo half-life of many biological molecules and was therefore applied to Cpl-1, a lysin specific for S. pneumoniae. Cysteine-specific PEGylation with either PEG 10K or 40K was achieved on Cpl-1 mutants, each containing an additional cysteine residue at different locations To the best of our knowledge, this is the first report of the PEGylation of bacteriophage lysin. Compared to the native enzyme, none of the PEGylated conjugates retained significant in vitro anti-pneumococcal lytic activity that would have justified further in vivo studies. Since the anti-microbial activity of the mutant enzymes used in this study was not affected by the introduction of the cysteine residue, our results implied that the presence of the PEG molecule was responsible for the inhibition. As most endolysins exhibit a similar modular structure, we believe that our work emphasizes the inability to improve the in vivo half-life of this class of enzybiotics using a cysteine-specific PEGylation strategy.