Use of the lambda Red recombinase system to rapidly generate mutants in Pseudomonas aeruginosa

<p>Abstract</p> <p>Background</p> <p>The Red recombinase system of bacteriophage lambda has been used to inactivate chromosomal genes in various bacteria and fungi. The procedure consists of electroporating a polymerase chain reaction (PCR) fragment that was obtained with a 1- or 3-step PCR protocol and that carries an antibiotic cassette flanked by a region homologous to the target locus into a strain that expresses the lambda Red recombination system.</p> <p>Results</p> <p>This system has been modified for use in <it>Pseudomonas aeruginosa</it>. Chromosomal DNA deletions of single genes were generated using 3-step PCR products containing flanking regions 400–600 nucleotides (nt) in length that are homologous to the target sequence. A 1-step PCR product with a homologous extension flanking region of only 100 nt was in some cases sufficient to obtain the desired mutant. We further showed that the <it>P. aeruginosa </it>strain PA14 non-redundant transposon library can be used in conjunction with the lambda Red technique to rapidly generate large chromosomal deletions or transfer mutated genes into various PA14 isogenic mutants to create multi-locus knockout mutants.</p> <p>Conclusion</p> <p>The lambda Red-based technique can be used efficiently to generate mutants in <it>P. aeruginosa</it>. The main advantage of this procedure is its rapidity as mutants can be easily obtained in less than a week if the 3-step PCR procedure is used, or in less than three days if the mutation needs to be transferred from one strain to another.</p

Pseudomonas aeruginosa Overrides the Virulence Inducing Effect of Opioids When It Senses an Abundance of Phosphate

The gut during critical illness represents a complex ecology dominated by the presence of healthcare associated pathogens, nutrient scarce conditions, and compensatory host stress signals. We have previously identified key environmental cues, opioids and phosphate depletion that independently activate the virulence of Pseudomonas aeruginosa. Opioids induce quinolone signal production (PQS), whereas phosphate depletion leads to a triangulated response between MvfR-PQS, pyoverdin, and phosphosensory/phosphoregulatory systems (PstS-PhoB). Yet how P. aeruginosa manages its response to opioids during nutrient scarce conditions when growth is limited and a quorum is unlikely to be achieved is important in the context of pathogenesis in gut during stress. To mimic this environment, we created nutrient poor conditions and exposed P. aeruginosa PAO1 to the specific k-opioid receptor agonist U-50,488. Bacterial cells exposed to the k-opioid expressed a striking increase in virulence- and multi-drug resistance-related genes that correlated to a lethal phenotype in C. elegans killing assays. Under these conditions, HHQ, a precursor of PQS, rather than PQS itself, became the main inducer for pqsABCDE operon expression. P. aeruginosa virulence expression in response to k-opioids required PqsE since ΔPqsE was attenuated in its ability to activate virulence- and efflux pumps-related genes. Extracellular inorganic phosphate completely changed the transcriptional response of PAO1 to the k- opioid preventing pqsABCDE expression, the activation of multiple virulence- and efflux pumps-related genes, and the ability of P. aeruginosa to kill C. elegans. These results indicate that when P. aeruginosa senses resource abundance in the form of phosphate, it overrides its response to compensatory host signals such as opioids to express a virulent and lethal phenotype. These studies confirm a central role for phosphate in P. aeruginosa virulence that might be exploited to design novel anti- virulence strategies

Evolution of metabolic divergence in <i>Pseudomonas aeruginosa</i> during long-term infection facilitates a proto-cooperative interspecies interaction

The effect of polymicrobial interactions on pathogen physiology and how it can act either to limit pathogen colonization or to potentiate pathogen expansion and virulence are not well understood. Pseudomonas aeruginosa and Staphylococcus aureus are opportunistic pathogens commonly found together in polymicrobial human infections. However, we have previously shown that the interactions between these two bacterial species are strain dependent. Whereas P. aeruginosa PAO1, a commonly used laboratory strain, effectively suppressed S. aureus growth, we observed a commensal-like interaction between the human host-adapted strain, DK2-P2M24-2003, and S. aureus. In this study, characterization by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) imaging mass spectrometry (IMS) and mass spectral (MS) molecular networking revealed a significant metabolic divergence between P. aeruginosa PAO1 and DK2-P2M24-2003, which comprised several virulence factors and signaling 4-hydroxy-2-alkylquinoline (HAQ) molecules. Strikingly, a further modulation of the HAQ profile was observed in DK2-P2M24-2003 during interaction with S. aureus, resulting in an area with thickened colony morphology at the P. aeruginosa–S. aureus interface. In addition, we found an HAQ-mediated protection of S. aureus by DK2-P2M24-2003 from the killing effect of tobramycin. Our findings suggest a model where the metabolic divergence manifested in human host-adapted P. aeruginosa is further modulated during interaction with S. aureus and facilitate a proto-cooperative P. aeruginosa–S. aureus relationship

Unravelling the genome-wide contributions of specific 2-alkyl-4-quinolones and PqsE to quorum sensing in Pseudomonas aeruginosa

The pqs quorum sensing (QS) system is crucial for Pseudomonas aeruginosa virulence both in vitro and in animal models of infection and is considered an ideal target for the development of anti-virulence agents. However, the precise role played by each individual component of this complex QS circuit in the control of virulence remains to be elucidated. Key components of the pqs QS system are 2-heptyl-4-hydroxyquinoline (HHQ), 2-heptyl-3-hydroxy-4-quinolone (PQS), 2-heptyl-4-hydroxyquinoline N-oxide (HQNO), the transcriptional regulator PqsR and the PQS-effector element PqsE. To define the individual contribution of each of these components to QS-mediated regulation, transcriptomic analyses were performed and validated on engineered P. aeruginosa strains in which the biosynthesis of 2-alkyl 4-quinolones (AQs) and expression of pqsE and pqsR have been uncoupled, facilitating the identification of the genes controlled by individual pqs system components. The results obtained demonstrate that i) the PQS biosynthetic precursor HHQ triggers a PqsR-dependent positive feedback loop that leads to the increased expression of only the pqsABCDE operon, ii) PqsE is involved in the regulation of diverse genes coding for key virulence determinants and biofilm development, iii) PQS promotes AQ biosynthesis, the expression of genes involved in the iron-starvation response and virulence factor production via PqsR-dependent and PqsR-independent pathways, and iv) HQNO does not influence transcription and hence does not function as a QS signal molecule. Overall this work has facilitated identification of the specific regulons controlled by individual pqs system components and uncovered the ability of PQS to contribute to gene regulation independent of both its ability to activate PqsR and to induce the iron-starvation response

Fitness of Isogenic Colony Morphology Variants of Pseudomonas aeruginosa in Murine Airway Infection

Chronic lung infections with Pseudomonas aeruginosa are associated with the diversification of the persisting clone into niche specialists and morphotypes, a phenomenon called ‘dissociative behaviour’. To explore the potential of P. aeruginosa to change its morphotype by single step loss-of–function mutagenesis, a signature-tagged mini-Tn5 plasposon library of the cystic fibrosis airway isolate TBCF10839 was screened for colony morphology variants under nine different conditions in vitro. Transposon insertion into 1% of the genome changed colony morphology into eight discernable morphotypes. Half of the 55 targets encode features of primary or secondary metabolism whereby quinolone production was frequently affected. In the other half the transposon had inserted into genes of the functional categories transport, regulation or motility/chemotaxis. To mimic dissociative behaviour of isogenic strains in lungs, pools of 25 colony morphology variants were tested for competitive fitness in an acute murine airway infection model. Six of the 55 mutants either grew better or worse in vivo than in vitro, respectively. Metabolic proficiency of the colony morphology variant was a key determinant for survival in murine airways. The most common morphotype of self-destructive autolysis did unexpectedly not impair fitness. Transposon insertions into homologous genes of strain PAO1 did not reproduce the TBCF10839 mutant morphotypes for 16 of 19 examined loci pointing to an important role of the genetic background on colony morphology. Depending on the chosen P. aeruginosa strain, functional genome scans will explore other areas of the evolutionary landscape. Based on our discordant findings of mutant phenotypes in P. aeruginosa strains PAO1, PA14 and TBCF10839, we conclude that the current focus on few reference strains may miss modes of niche adaptation and dissociative behaviour that are relevant for the microevolution of complex traits in the wild

Ancillary human health benefits of improved air quality resulting from climate change mitigation

<p>Abstract</p> <p>Background</p> <p>Greenhouse gas (GHG) mitigation policies can provide ancillary benefits in terms of short-term improvements in air quality and associated health benefits. Several studies have analyzed the ancillary impacts of GHG policies for a variety of locations, pollutants, and policies. In this paper we review the existing evidence on ancillary health benefits relating to air pollution from various GHG strategies and provide a framework for such analysis.</p> <p>Methods</p> <p>We evaluate techniques used in different stages of such research for estimation of: (1) changes in air pollutant concentrations; (2) avoided adverse health endpoints; and (3) economic valuation of health consequences. The limitations and merits of various methods are examined. Finally, we conclude with recommendations for ancillary benefits analysis and related research gaps in the relevant disciplines.</p> <p>Results</p> <p>We found that to date most assessments have focused their analysis more heavily on one aspect of the framework (e.g., economic analysis). While a wide range of methods was applied to various policies and regions, results from multiple studies provide strong evidence that the short-term public health and economic benefits of ancillary benefits related to GHG mitigation strategies are substantial. Further, results of these analyses are likely to be underestimates because there are a number of important unquantified health and economic endpoints.</p> <p>Conclusion</p> <p>Remaining challenges include integrating the understanding of the relative toxicity of particulate matter by components or sources, developing better estimates of public health and environmental impacts on selected sub-populations, and devising new methods for evaluating heretofore unquantified and non-monetized benefits.</p

Mechanistic analysis of a synthetic inhibitor of the LasI quorum-sensing signal synthase

Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen responsible for many human infections. LasI is an acyl-homoserine lactone synthase that produces a quorum-sensing (QS) signal that positively regulates numerous P. aeruginosa virulence determinants. The inhibition of the LasI protein is therefore an attractive drug target. In this study, a novel in silico to in vitro complementation was applied to screen thiazolidinedione-type compounds for their ability to inhibit biofilm formation at concentrations not affecting bacterial growth. The compound (z)-5-octylidenethiazolidine-2, 4-dione (TZD-C8) was a strong inhibitor of biofilm formation and chosen for further study. Structural exploration of in silico docking predicted that the compound had high affinity for the LasI activity pocket. The TZD-C8 compound was also predicted to create hydrogen bonds with residues Arg30 and Ile107. Site-directed mutagenesis (SDM) of these two sites demonstrated that TZD-C8 inhibition was abolished in the lasI double mutant PAO-R30D, I107S. In addition, in vitro swarming motility and quorum sensing signal production were affected by TZD-C 8, confirming this compound alters the cell to cell signalling circuitry. Overall, this novel inhibitor of P. aeruginosa quorum sensing shows great promise and validates our mechanistic approach to discovering inhibitors of LuxI-type acyl-homoserine lactone synthases