Inhibition of PQS signaling by the Pf bacteriophage protein PfsE enhances viral replication in Pseudomonas aeruginosa.

Quorum sensing, a bacterial signaling system that coordinates group behaviors as a function of cell density, plays an important role in regulating viral (phage) defense mechanisms in bacteria. The opportunistic pathogen Pseudomonas aeruginosa is a model system for the study of quorum sensing. P. aeruginosa is also frequently infected by Pf prophages that integrate into the host chromosome. Upon induction, Pf phages suppress host quorum sensing systems; however, the physiological relevance and mechanism of suppression are unknown. Here, we identify the Pf phage protein PfsE as an inhibitor of Pseudomonas Quinolone Signal (PQS) quorum sensing. PfsE binds to the host protein PqsA, which is essential for the biosynthesis of the PQS signaling molecule. Inhibition of PqsA increases the replication efficiency of Pf virions when infecting a new host and when the Pf prophage switches from lysogenic replication to active virion replication. In addition to inhibiting PQS signaling, our prior work demonstrates that PfsE also binds to PilC and inhibits type IV pili extension, protecting P. aeruginosa from infection by type IV pili-dependent phages. Overall, this work suggests that the simultaneous inhibition of PQS signaling and type IV pili by PfsE may be a viral strategy to suppress host defenses to promote Pf replication while at the same time protecting the susceptible host from competing phages.

Exploring fluorinated heptose phosphate analogues as inhibitors of HldA and HldE, key enzymes in the biosynthesis of lipopolysaccharide.

The growing threat of bacterial resistance to antibiotics has led to the rise of anti-virulence strategies as a promising approach. These strategies aim to disarm bacterial pathogens and improve their clearance by the host immune system. Lipopolysaccharide, a key virulence factor in Gram-negative bacteria, has been identified as a potential target for anti-virulence agents. In this study, we focus on inhibiting HldA and HldE, bacterial enzymes from the heptose biosynthesis pathway, which plays a key role in lipopolysaccharide biosynthesis. We present the synthesis of two fluorinated non-hydrolysable heptose phosphate analogues. Additionally, the inhibitory activity of a family of eight heptose phosphate analogues against HldA and HldE was assessed. This evaluation revealed inhibitors with affinities in the low µM range, with the most potent compound showing inhibition constant values of 15.4 µM for HldA and 16.9 µM for HldE. The requirement for a phosphate group at the C-7 position was deemed essential for inhibitory activity, while the presence of a hydroxy anomeric group was found to be beneficial, a phenomenon rationalized through computational modeling. Additionally, the introduction of a single fluorine atom α to the phosphonate moiety conferred a slight advantage for inhibition. These findings suggest that mimicking the structure of d-glycero-ß-d-manno-heptose 1,7-bisphosphate, the product of the phosphorylation step in heptose biosynthesis, could be a promising strategy to disrupt this biosynthetic pathway. In terms of the in vivo effects, these heptose phosphate analogues neither demonstrated significant LPS-disrupting effects nor exhibited growth inhibitory activity on their own. Additionally, they did not alter the susceptibility of bacteria to hydrophobic antibiotics. The highly charged nature of these molecules may hinder their ability to penetrate the bacterial cell wall. To overcome this limitation, alternative strategies such as incorporating protecting groups that facilitate their entry and can subsequently be cleaved within the bacterial cytoplasm could be explored.

Pseudomonas aeruginosa Strains from Both Clinical and Environmental Origins Readily Adopt a Stable Small-Colony-Variant Phenotype Resulting from Single Mutations in c-di-GMP Pathways.

A subpopulation of small-colony variants (SCVs) is a frequently observed feature of Pseudomonas aeruginosa isolates obtained from colonized cystic fibrosis lungs. Since most SCVs have until now been isolated from clinical samples, it remains unclear how widespread the ability of P. aeruginosa strains to develop this phenotype is and what the genetic mechanism(s) behind the emergence of SCVs are according to the origin of the isolate. In the present work, we investigated the ability of 22 P. aeruginosa isolates from various environmental origins to spontaneously adopt an SCV-like smaller alternative morphotype distinguishable from that of the ancestral parent strain under laboratory culture conditions. We found that all the P. aeruginosa strains tested could adopt an SCV phenotype, regardless of their origin. Whole-genome sequencing of SCVs obtained from clinical and environmental sources revealed single mutations exclusively in two distinct c-di-GMP signaling pathways, the Wsp and YfiBNR pathways. We conclude that the ability to switch to an SCV phenotype is a conserved feature of P. aeruginosa and results from the acquisition of a stable genetic mutation, regardless of the origin of the strain. IMPORTANCE P. aeruginosa is an opportunistic pathogen that thrives in many environments. It poses a significant health concern, notably because this bacterium is the most prevalent pathogen found in the lungs of people with cystic fibrosis. In infected hosts, its persistence is considered related to the emergence of an alternative small-colony-variant (SCV) phenotype. By reporting the distribution of P. aeruginosa SCVs in various nonclinical environments and the involvement of c-di-GMP in SCV emergence from both clinical and environmental strains, this work contributes to understanding a conserved adaptation mechanism used by P. aeruginosa to adapt readily in all environments. Hindering this adaptation strategy could help control persistent infection by P. aeruginosa.

Pseudomonas aeruginosa isolates defective in function of the LasR quorum sensing regulator are frequent in diverse environmental niches.

The saprophyte Pseudomonas aeruginosa is a versatile opportunistic pathogen causing infections in immunocompromised individuals. To facilitate its adaptation to a large variety of niches, this bacterium exploits population density-dependent gene regulation systems called quorum sensing (QS). In P. aeruginosa, three distinct but interrelated QS systems (las, rhl and pqs) regulate the production of many survival and virulence functions. In prototypical strains, the las system, through its transcriptional regulator LasR, is important for the full activation of the rhl and pqs systems. Still, LasR-deficient isolates have been reported, mostly sampled from the lungs of people with cystic fibrosis, where they are considered selected by the chronic infection environment. In this study, we show that a defect in LasR activity appears to be an actually widespread mechanism of adaptation in this bacterium. Indeed, we found abundant LasR-defective isolates sampled from hydrocarbon-contaminated soils, hospital sink drains and meat/fish market environments, using an approach based on phenotypic profiling, supported by gene sequencing. Interestingly, several LasR-defective isolates maintain an active rhl system or are deficient in pqs system signalling. The high prevalence of a LasR-defective phenotype among environmental P. aeruginosa isolates questions the role of QS in niche adaptation.

Social cheating in a Pseudomonas aeruginosa quorum-sensing variant.

The opportunistic bacterial pathogen Pseudomonas aeruginosa has a layered acyl-homoserine lactone (AHL) quorum-sensing (QS) system, which controls production of a variety of extracellular metabolites and enzymes. The LasRI system activates genes including those coding for the extracellular protease elastase and for the second AHL QS system, RhlRI. Growth of P. aeruginosa on casein requires elastase production and LasR-mutant social cheats emerge in populations growing on casein. P. aeruginosa colonizes the lungs of individuals with the genetic disease cystic fibrosis (CF), and LasR mutants can be isolated from the colonized lungs; however, unlike laboratory-generated LasR mutants, many of these CF isolates have functioning RhlR-RhlI systems. We show that one such mutant can use the RhlR-RhlI system to activate expression of elastase and grow on casein. We carried out social-evolution experiments by growing this isolate on caseinate and, as with wild-type P. aeruginosa, elastase-negative mutants emerge as cheats, but these are not RhlR mutants; rather, they are mutants that do not produce the non-AHL Pseudomonas quinolone signal (PQS). Furthermore, we generated a RhlRI mutant and showed it had a fitness defect when growing together with the parent. Apparently, RhlR QS and PQS collude to support growth on caseinate in the absence of a functional LasR. Our findings provide a plausible explanation as to why P. aeruginosa LasR mutants, but not RhlR mutants, are common in CF lungs.

Total Synthesis of a Chimeric Glycolipid Bearing the Partially Acetylated Backbone of Sponge-Derived Agminoside E.

We describe the total synthesis of a chimeric glycolipid bearing both the partially acetylated backbone of sponge-derived agminoside E and the (R)-3-hydroxydecanoic acid chain of bacterial rhamnolipids. The branched pentaglucolipid skeleton was achieved using a [3 + 2] disconnection approach. The ß-(1 → 2) and ß-(1 → 4)-glycosidic bonds were synthesized through a combination of NIS/Yb(OTf)3- and TMSOTf-mediated stereoselective glycosylations of thiotolyl, N-phenyltrifluoroacetimidate, and trichloroacetimidate donors. Late-stage pentaacetylation, Staudinger reduction of a (2-azidomethyl)benzoyl group, followed by continuous-flow microfluidic hydrogenolysis completed the total synthesis of the structurally simplified glycolipid, whose partial acetylation pattern on the glycan part was identical to agminoside E. Our study lays the foundation for the total synthesis of sponge-derived agminosides and the understanding of their biological functions in sponges.

ScmR, a Global Regulator of Gene Expression, Quorum Sensing, pH Homeostasis, and Virulence in Burkholderia thailandensis.

The nonpathogenic soil saprophyte Burkholderia thailandensis is a member of the Burkholderia pseudomallei/B. thailandensis/B. mallei group, which also comprises the closely related human pathogens B. pseudomallei and Burkholderia mallei responsible for the melioidosis and glanders diseases, respectively. ScmR, a recently identified LysR-type transcriptional regulator in B. thailandensis, acts as a global transcriptional regulator throughout the stationary phase and modulates the production of a wide range of secondary metabolites, including N-acyl-l-homoserine lactones and 4-hydroxy-3-methyl-2-alkylquinolines and virulence in the Caenorhabditis elegans nematode worm host model, as well as several quorum sensing (QS)-dependent phenotypes. We have investigated the role of ScmR in B. thailandensis strain E264 during the exponential phase. We used RNA sequencing transcriptomic analyses to identify the ScmR regulon, which was compared to the QS-controlled regulon, showing a considerable overlap between the ScmR-regulated genes and those controlled by QS. We characterized several genes modulated by ScmR using quantitative reverse transcription-PCR or mini-CTX-lux transcriptional reporters, including the oxalate biosynthetic gene obc1 required for pH homeostasis, the orphan LuxR-type transcriptional regulator BtaR5-encoding gene, and the bsa (Burkholderia secretion apparatus) type III secretion system genes essential for both B. pseudomallei and B. mallei pathogenicity, as well as the scmR gene itself. We confirmed that the transcription of scmR is under QS control, presumably ensuring fine-tuned modulation of gene expression. Finally, we demonstrated that ScmR influences virulence using the fruit fly model host Drosophila melanogaster We conclude that ScmR represents a central component of theB. thailandensis QS regulatory network.IMPORTANCE Coordination of the expression of genes associated with bacterial virulence and environmental adaptation is often dependent on quorum sensing (QS). The QS circuitry of the nonpathogenic bacterium Burkholderia thailandensis, widely used as a model system for the study of the human pathogen Burkholderia pseudomallei, is complex. We found that the LysR-type transcriptional regulator, ScmR, which is highly conserved and involved in the control of virulence/survival factors in the Burkholderia genus, is a global regulator mediating gene expression through the multiple QS systems coexisting in B. thailandensis, as well as QS independently. We conclude that ScmR represents a key QS modulatory network element, ensuring tight regulation of the transcription of QS-controlled genes, particularly those required for acclimatization to the environment.

Swarming motility growth favours the emergence of a subpopulation of Pseudomonas aeruginosa quorum-sensing mutants.

Pseudomonas aeruginosa exploits several types of motility behaviours to colonize diverse environments. One of these is swarming motility, a coordinated group movement on a semi-solid surface. This bacterium needs to express a functional flagellum and produce rhamnolipids to display this type of social motility. A ΔhptB mutant, a gene part of the Gac/Rsm signalling pathway, produces rhamnolipids and expresses a functional flagellum but has an important swarming defect. Experimental-directed evolution was performed on this mutant under swarming conditions to obtain compensatory mutations and thus identify genes responsible for its deficient swarming phenotype. Unexpectedly, a gain-of-function subpopulation emerged from this evolution with mutations in lasR, which codes for a key quorum-sensing transcriptional regulator. Furthermore, we found that lasR- mutants even emerge at high frequencies in the wild-type strain when using the same experimental evolution strategy. The resulting evolved population, largely composed of LasR-defective mutants, is fitter than the original strain in swarming motility. We also established that lasR- mutants have a growth advantage under swarming conditions when compared with wild-type. Our results demonstrate that a social phenotype, that is, swarming motility, favours the emergence of mutants deficient in a quorum-sensing regulatory pathway to the benefit of the whole population.

Cationic RuII Cyclopentadienyl Complexes with Antifungal Activity against Several Candida Species.

Fungal infections, including those caused by antifungal-resistant Candida, are a very challenging health problem worldwide. Whereas different ruthenium complexes were previously studied for their anti-Candida potential, Ru-cyclopentadienyl complexes were overlooked. Here, we report an antifungal activity assessment of three Ru-cyclopentadienyl complexes with some insights into their potential mode of action. Among these complexes, only the cationic species [Ru-ACN]+ and [Ru-ATZ]+ displayed a significant antifungal activity against different Candida strains, notably against the ones that did not respond to one of the most currently used antifungal drugs fluconazole (FCZ). However, no apparent activity was observed for the neutral species, Ru-Cl, thus indicating the important role of the cationic backbone of these complexes in their biological activity. We suggest that reactive oxygen species (ROS) generation might be involved in the mechanism of action of these complexes as, unlike neutral Ru-Cl, [Ru-ACN]+ and [Ru-ATZ]+ could generate intracellular concentration-dependent ROS. We also observed a correlation between the ruthenium cellular uptake, ROS generation and fungal growth inhibitory activity of the compounds. Furthermore, docking simulations showed that the CYP51 enzyme can form more energetically favorable complexes with [Ru-ATZ]+ than fluconazole (FCZ); this suggests that CYP51 inhibition could also be considered as a potential mode of action.

Burkholderia thailandensis Methylated Hydroxyalkylquinolines: Biosynthesis and Antimicrobial Activity in Cocultures.

The bacterium Burkholderia thailandensis produces an arsenal of secondary metabolites that have diverse structures and roles in the ecology of this soil-dwelling bacterium. In coculture experiments, B. thailandensis strain E264 secretes an antimicrobial that nearly eliminates another soil bacterium, Bacillus subtilis strain 168. To identify the antimicrobial, we used a transposon mutagenesis approach. This screen identified antimicrobial-defective mutants with insertions in the hmqA, hmqC, and hmqF genes involved in biosynthesis of a family of 2-alkyl-4(1H)-quinolones called 4-hydroxy-3-methyl-2-alkenylquinolines (HMAQs), which are closely related to the Pseudomonas aeruginosa 4-hydroxy-2-alkylquinolines (HAQs). Insertions also occurred in the previously uncharacterized gene BTH_II1576 ("hmqL"). The results confirm that BTH_II1576 is involved in generating N-oxide derivatives of HMAQs (HMAQ-NOs). Synthetic HMAQ-NO is active against B. subtilis 168, showing ∼50-fold more activity than HMAQ. Both the methyl group and the length of the carbon side chain account for the high activity of HMAQ-NO. The results provide new information on the biosynthesis and activities of HMAQs and reveal new insight into how these molecules might be important for the ecology of B. thailandensisIMPORTANCE The soil bacterium Burkholderia thailandensis produces 2-alkyl-4(1H)-quinolones that are mostly methylated 4-hydroxyalkenylquinolines, a family of relatively unstudied metabolites similar to molecules also synthesized by Pseudomonas aeruginosa Several of the methylated 4-hydroxyalkenylquinolines have antimicrobial activity against other species. We show that Bacillus subtilis strain 168 is particularly susceptible to N-oxidated methylalkenylquinolines (HMAQ-NOs). We confirmed that HMAQ-NO biosynthesis requires the previously unstudied protein HmqL. These results provide new information about the biology of 2-alkyl-4(1H)-quinolones, particularly the methylated 4-hydroxyalkenylquinolines, which are unique to B. thailandensis This study also has importance for understanding B. thailandensis secondary metabolites and has implications for potential therapeutic development.

Synthesis and Antimicrobial Activity of Burkholderia-Related 4-Hydroxy-3-methyl-2-alkenylquinolines (HMAQs) and Their N-Oxide Counterparts.

The Burkholderia genus offers a promising potential in medicine because of the diversity of biologically active natural products encoded in its genome. Some pathogenic Burkholderia spp. biosynthesize a specific class of antimicrobial 2-alkyl-4(1H)-quinolones, i.e., 4-hydroxy-3-methyl-2-alkenylquinolines (HMAQs) and their N-oxide derivatives (HMAQNOs). Herein, we report the synthesis of a series of six HMAQs and HMAQNOs featuring a trans-Δ2 double bond at the C2-alkyl chain. The quinolone scaffold was obtained via the Conrad-Limpach approach, while the (E)-2-alkenyl chain was inserted through Suzuki-Miyaura cross-coupling under microwave radiation without noticeable isomerization according to the optimized conditions. Subsequent oxidation of enolate-protected HMAQs cleanly led to the formation of HMAQNOs following cleavage of the ethyl carbonate group. Synthetic HMAQs and HMAQNOs were evaluated in vitro for their antimicrobial activity against different Gram-negative and Gram-positive bacteria as well as against molds and yeasts. The biological results support and extend the potential of HMAQs and HMAQNOs as antimicrobials, especially against Gram-positive bacteria. We also confirm the involvement of HMAQs in the autoregulation of the Hmq system in Burkholderia ambifaria.

Phenylacetyl Coenzyme A, Not Phenylacetic Acid, Attenuates CepIR-Regulated Virulence in Burkholderia cenocepacia.

During phenylalanine catabolism, phenylacetic acid (PAA) is converted to phenylacetyl coenzyme A (PAA-CoA) by a ligase, PaaK, and then PAA-CoA is epoxidized by a multicomponent monooxygenase, PaaABCDE, before further degradation through the tricarboxylic acid (TCA) cycle. In the opportunistic pathogen Burkholderia cenocepacia, loss of paaABCDE attenuates virulence factor expression, which is under the control of the LuxIR-like quorum sensing (QS) system, CepIR. To further investigate the link between CepIR-regulated virulence and PAA catabolism, we created knockout mutants of the first step of the pathway (PAA-CoA synthesis by PaaK) and characterized them in comparison to a paaABCDE mutant using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and virulence assays. We found that while loss of PaaABCDE decreased virulence, deletion of the paaK genes resulted in a more virulent phenotype than that of the wild-type strain. Deletion of either paaK or paaABCDE led to higher levels of released PAA but no differences in levels of internal accumulation compared to the wild-type level. While we found no evidence of direct cepIR downregulation by PAA-CoA or PAA, a low-virulence cepR mutant reverted to a virulent phenotype upon removal of the paaK genes. On the other hand, removal of paaABCDE in the cepR mutant did not impact its attenuated phenotype. Together, our results suggest an indirect role for PAA-CoA in suppressing B. cenocepacia CepIR-activated virulence.IMPORTANCE The opportunistic pathogen Burkholderia cenocepacia uses a chemical signal process called quorum sensing (QS) to produce virulence factors. In B. cenocepacia, QS relies on the presence of the transcriptional regulator CepR which, upon binding QS signal molecules, activates virulence. In this work, we found that even in the absence of CepR, B. cenocepacia can elicit a pathogenic response if phenylacetyl-CoA, an intermediate of the phenylacetic acid degradation pathway, is not produced. Instead, accumulation of phenylacetyl-CoA appears to attenuate pathogenicity. Therefore, we have discovered that it is possible to trigger virulence in the absence of CepR, challenging the classical view of activation of virulence by this QS mechanism. Our work provides new insight into the relationship between metabolism and virulence in opportunistic bacteria. We propose that in the event that QS signaling molecules cannot accumulate to trigger a pathogenic response, a metabolic signal can still activate virulence in B. cenocepacia.

Aspergillus-Pseudomonas interaction, relevant to competition in airways.

In airways of immunocompromised patients and individuals with cystic fibrosis, Pseudomonas aeruginosa and Aspergillus fumigatus are the most common opportunistic bacterial and fungal pathogens. Both pathogens form biofilms and cause acute and chronic illnesses. Previous studies revealed that P. aeruginosa is able to inhibit A. fumigatus biofilms in vitro. While numerous P. aeruginosa molecules have been shown to affect A. fumigatus, there never has been a systematic approach to define the principal causative agent. We studied 24 P. aeruginosa mutants, with deletions in genes important for virulence, iron acquisition, or quorum sensing, for their ability to interfere with A. fumigatus biofilms. Cells, planktonic or biofilm culture filtrates of four P. aeruginosa mutants, pvdD-pchE-, pvdD-, lasR-rhlR-, and lasR-, inhibited A. fumigatus biofilm metabolism or planktonic A. fumigatus growth significantly less than P. aeruginosa wild type. The common defect of these four mutants was a lack in the production of the P. aeruginosa siderophore pyoverdine. Pure pyoverdine affected A. fumigatus biofilm metabolism, and restored inhibition by the above mutants. In lungs from cystic fibrosis patients, pyoverdine production and antifungal activity correlated. The key inhibitory mechanism for pyoverdine was iron-chelation and denial of iron to A. fumigatus. Further experiments revealed a counteracting, self-protective mechanism by A. fumigatus, based on A. fumigatus siderophore production.

Two rsaM Homologues Encode Central Regulatory Elements Modulating Quorum Sensing in Burkholderia thailandensis.

The bacterium Burkholderia thailandensis possesses three N-acyl-l-homoserine lactone (AHL) quorum sensing (QS) systems designated BtaI1/BtaR1 (QS-1), BtaI2/BtaR2 (QS-2), and BtaI3/BtaR3 (QS-3). These QS systems are associated with the biosynthesis of N-octanoyl-homoserine lactone (C8-HSL), N-3-hydroxy-decanoyl-homoserine lactone (3OHC10-HSL), and N-3-hydroxy-octanoyl-homoserine lactone (3OHC8-HSL), which are produced by the LuxI-type synthases BtaI1, BtaI2, and BtaI3 and modulated by the LuxR-type transcriptional regulators BtaR1, BtaR2, and BtaR3. The btaR1-btaI1 and btaR2-btaI2 gene clusters each carry an additional gene encoding a homologue of the QS repressor RsaM originally identified in the phytopathogen Pseudomonas fuscovaginae and thus here named rsaM1 and rsaM2, respectively. We have characterized the functions of these two conserved rsaM homologues and demonstrated their involvement in the regulation of AHL biosynthesis in B. thailandensis strain E264. We quantified the production of C8-HSL, 3OHC10-HSL, and 3OHC8-HSL by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) in the wild-type strain and in the rsaM1 and rsaM2 mutants, and we monitored btaI1, btaI2, and btaI3 expression using chromosomal mini-CTX-lux transcriptional reporters. The transcription of btaR1, btaR2, and btaR3 was also measured by quantitative reverse transcription-PCR (qRT-PCR). We observed that RsaM1 mainly represses the QS-1 system, whereas RsaM2 principally represses the QS-2 system. We also found that both rsaM1 and rsaM2 are QS controlled and negatively autoregulated. We conclude that RsaM1 and RsaM2 are an integral part of the QS circuitry of B. thailandensis and play a major role in the hierarchical and homeostatic organization of the QS-1, QS-2, and QS-3 systems.IMPORTANCE Quorum sensing (QS) is commonly involved in the coordination of gene transcription associated with the establishment of host-pathogen interactions and acclimatization to the environment. We present the functional characterization of two rsaM homologues in the regulation of the multiple QS systems coexisting in the nonpathogenic bacterium Burkholderia thailandensis, which is widely used as a model system for the study of the human pathogen Burkholderia pseudomallei We found that inactivation of these rsaM homologues, which are clustered with the other QS genes, profoundly affects the QS circuitry of B. thailandensis We conclude that they constitute essential regulatory components of the QS modulatory network and provide additional layers of regulation to modulate the transcription of QS-controlled genes, particularly those linked to environmental adaptation.

Studies of Pseudomonas aeruginosa Mutants Indicate Pyoverdine as the Central Factor in Inhibition of Aspergillus fumigatus Biofilm.

Pseudomonas aeruginosa and Aspergillus fumigatus are common opportunistic bacterial and fungal pathogens, respectively. They often coexist in airways of immunocompromised patients and individuals with cystic fibrosis, where they form biofilms and cause acute and chronic illnesses. Hence, the interactions between them have long been of interest and it is known that P. aeruginosa can inhibit A. fumigatusin vitro We have approached the definition of the inhibitory P. aeruginosa molecules by studying 24 P. aeruginosa mutants with various virulence genes deleted for the ability to inhibit A. fumigatus biofilms. The ability of P. aeruginosa cells or their extracellular products produced during planktonic or biofilm growth to affect A. fumigatus biofilm metabolism or planktonic A. fumigatus growth was studied in agar and liquid assays using conidia or hyphae. Four mutants, the pvdD pchE, pvdD, lasR rhlR, and lasR mutants, were shown to be defective in various assays. This suggested the P. aeruginosa siderophore pyoverdine as the key inhibitory molecule, although additional quorum sensing-regulated factors likely contribute to the deficiency of the latter two mutants. Studies of pure pyoverdine substantiated these conclusions and included the restoration of inhibition by the pyoverdine deletion mutants. A correlation between the concentration of pyoverdine produced and antifungal activity was also observed in clinical P. aeruginosa isolates derived from lungs of cystic fibrosis patients. The key inhibitory mechanism of pyoverdine was chelation of iron and denial of iron to A. fumigatusIMPORTANCE Interactions between human pathogens found in the same body locale are of vast interest. These interactions could result in exacerbation or amelioration of diseases. The bacterium Pseudomonas aeruginosa affects the growth of the fungus Aspergillus fumigatus Both pathogens form biofilms that are resistant to therapeutic drugs and host immunity. P. aeruginosa and A. fumigatus biofilms are found in vivo, e.g., in the lungs of cystic fibrosis patients. Studying 24 P. aeruginosa mutants, we identified pyoverdine as the major anti-A. fumigatus compound produced by P. aeruginosa Pyoverdine captures iron from the environment, thus depriving A. fumigatus of a nutrient essential for its growth and metabolism. We show how microbes of different kingdoms compete for essential resources. Iron deprivation could be a therapeutic approach to the control of pathogen growth.

Cyclic-di-GMP levels affect Pseudomonas aeruginosa fitness in the presence of imipenem.

A large number of genes coding for enzymes predicted to synthesize and degrade 3'-5'-cyclic diguanylic acid (c-di-GMP) is found in most bacterial genomes and this dinucleotide emerged as an intracellular signal-controlling bacterial behaviour. An association between high levels of c-di-GMP and antibiotic resistance may be expected because c-di-GMP regulates biofilm formation and this mode of growth leads to enhanced antibiotic resistance. However, a clear understanding of this correlation has not been established. We found that increased levels of c-di-GMP in Pseudomonas aeruginosa improve fitness in the presence of imipenem, even when grown as planktonic cells. P. aeruginosa post-transcriptionally regulates the amounts of five porins in response to c-di-GMP, including OprD, responsible for imipenem uptake. Cells with low c-di-GMP levels are consequently more sensitive to this antibiotic. Main efflux pumps or ß-lactamase genes did not show altered mRNA levels in P. aeruginosa strains with modified different c-di-GMP concentrations. Together, our findings show that c-di-GMP levels modulate fitness of planktonic cultures in the presence of imipenem.

Cyclic-di-GMP levels affect Pseudomonas aeruginosa fitness in the presence of imipenem.

A large number of genes coding for enzymes predicted to synthesize and degrade 3'-5-cyclic diguanylic acid (c-di-GMP) is found in most bacterial genomes and this dinucleotide emerged as an intracellular signal-controlling bacterial behaviour. An association between high levels of c-di-GMP and antibiotic resistance may be expected because c-di-GMP regulates biofilm formation and this mode of growth leads to enhanced antibiotic resistance. However, a clear understanding of this correlation has not been established. We found that increased levels of c-di-GMP in Pseudomonas aeruginosa improve fitness in the presence of imipenem, even when grown as planktonic cells. P. aeruginosa post-transcriptionally regulates the amounts of five porins in response to c-di-GMP, including OprD, responsible for imipenem uptake. Cells with low c-di-GMP levels are consequently more sensitive to this antibiotic. Main efflux pumps or ß-lactamase genes did not show altered mRNA levels in P. aeruginosa strains with modified different c-di-GMP concentrations. Together, our findings show that c-di-GMP levels modulate fitness of planktonic cultures in the presence of imipenem.

Structural characterization of a nonionic rhamnolipid from Burkholderia lata.

We present the isolation and structural characterization of a novel nonionic dirhamnolipid methyl ester produced by the bacterium Burkholderia lata. The structure and the absolute configuration of the isolated dirhamnolipid bearing a symmetrical C14-C14 methyl ester chain were thoroughly investigated through chemical degradation and spectroscopic methods including 1D and 2D NMR analysis, HR-ESI-TOF-MS, chiral GC-MS, and polarimetry. Our work represents the first mention in the literature of a rhamnolipid methyl ester from Burkholderia species.

The end of the reign of a "master regulator''? A defect in function of the LasR quorum sensing regulator is a common feature of Pseudomonas aeruginosa isolates.

Pseudomonas aeruginosa, a bacterium causing infections in immunocompromised individuals, regulates several of its virulence functions using three interlinked quorum sensing (QS) systems (las, rhl, and pqs). Despite its presumed importance in regulating virulence, dysfunction of the las system regulator LasR occurs frequently in strains isolated from various environments, including clinical infections. This newfound abundance of LasR-defective strains calls into question existing hypotheses regarding their selection. Indeed, current assumptions concerning factors driving the emergence of LasR-deficient isolates and the role of LasR in the QS hierarchy must be reconsidered. Here, we propose that LasR is not the primary master regulator of QS in all P. aeruginosa genetic backgrounds, even though it remains ecologically significant. We also revisit and complement current knowledge on the ecology of LasR-dependent QS in P. aeruginosa, discuss the hypotheses explaining the putative adaptive benefits of selecting against LasR function, and consider the implications of this renewed understanding.

Vfm a new quorum sensing system controls the virulence of Dickeya dadantii.

Dickeya dadantii is a plant pathogen that secretes cell wall-degrading enzymes (CWDE) that are responsible for soft-rot symptoms. Virulence genes are expressed in a concerted manner and culminate when bacterial multiplication slows. We identify a 25 kb vfm cluster required for D. dadantii CWDE production and pathogenesis. The vfm cluster encodes proteins displaying similarities both with enzymes involved in amino acid activation and with enzymes involved in fatty acid biosynthesis. These similarities suggest that the vfm genes direct the production of a metabolite. Cell-free supernatant from the D. dadantii wild-type strain restores CWDE production in vfm mutants. Collectively, our results indicate that vfm genes direct the synthesis of an extracellular signal and constitute a new quorum sensing system. Perception of the signal is achieved by the two-component system VfmH-VfmI, which activates the expression of the vfmE gene encoding an AraC regulator. VfmE then activates both the transcription of the CWDE genes and the expression of the vfm operons. The vfm gene cluster does not seem to be widespread among bacterial species but is conserved in other Dickeya species and could have been laterally transferred to Rahnella. This work highlights that entirely new families of bacterial languages remain to be discovered.