New approaches to detect and inhibit quorum sensing activity in Pseudomonas aeruginosa

Pseudomonas aeruginosa (PA), a Gram-negative opportunistic rod with ubiquitous presence in a panoply of different environments, secretes a wide array of virulence determinants that have established it as one of the leading nosocomial pathogens. Many of these virulence factors are regulated by the quorum sensing (QS) system that responds to environmental cell density variations. PA can ultimately trigger the onset of severe acute and chronic infections, especially in immunosuppressed subjects. The QS network in PA is comprised of at least four multi-layered interconnected subsystems with hierarchical organisation. From these, three (las, rhl and pqs) play a pivotal role in the production of virulence factors (e.g., lectins and pyocyanin) with relevant participation in the development and maintenance of biofilm matrices. The QS network is divided in two major signaling pathways, the one driven by N-acylhomoserine lactone signals and the one driven by 2-alkyl-4-quinolone molecules. Two alkyl quinolones of core importance exist in PA, the 2-heptyl-3-hydroxy-4(1H)quinolone, typically recognised as the “Pseudomonas quinolone signal” (PQS) and its precursor 2-heptyl-4(1H)-quinolone (HHQ). In addition to the regulatory involvement of the las and rhl quorum sensing systems, the biosynthesis of PQS production is also positively regulated by PqsR-dependent transcription of the pqsABCDE operon (a multivirulence factor regulator also known as MvfR). For that reason, the alkyl quinolone (AQ) signalling pathway, and more specifically its major regulator PqsR, are widely seen as promising targets for novel antimicrobial approaches. Because of the growing presence of multidrug resistant PA in the clinical setting, representing both an immediate menace to immunocompromised patients and a heavy burden on hospital budgets, the development of more rapid and affordable screening strategies for detection of the pathogen are required. Thus, new screening strategies adapted to clinical samples and successful novel synthetic small PqsR antagonists can lead the way to a new Era in the battle against hyper-virulent/-resistant PA strains. Primarily focusing on the AQ system, this research project investigated three inter-related areas, namely: (a) the highthroughput screening of novel synthetic small molecule antagonists of the PqsR protein, designed for suppression of virulence-associated phenotypes, (b) the development of a luminescent PQS-based screening bioreporter to be applied in the clinical setting, and finally (c) the optimisation of a methodology combining liquid extraction surface analysis (LESA) and mass spectrometry (MS) for screening PQS-related AQs from in vivo bacterial extracts, and also intended for future screening of a variety of clinical samples (e.g., blood plasma, urine and saliva). A large number of synthetic small molecules with putative PqsR antagonism were obtained from our French partner GreenPharma, and studied for their capacity to interfere with expression of the key player of the pqs system, PqsR. By applying a rational selection strategy, based on the inhibitory effects on pqsA expression, assessment of metabolical exertion, and assay studies on the ultimate repression of key virulence-associated phenotypes (lectins LecA and LecB, pyocyanin, PQS-associated AQs) and impact on biolfilm formation, a final selection comprised of the four best antagonists was obtained. Compounds GPZ002966, GPZ004927, GPZ824390 and GPZ273902 had their cytotoxicity subsequently studied keeping in mind their applicability in pre-clinical studies. Overall, these PqsR antagonists promoted very strong inhibition of pqsA and lecA expression, strongly reduced production of pyocyanin and PQS-related AQs (HHQ, HQNO and C7-PQS itself), showed a strong degree of biofilm inhibition, with IC50 scores sitting at the nanomolar level, and no signs of metabolical arrest was reported by the test strains used. Some explanations, focusing on the functional and structural organisation/composition of these compounds are also offered based on a comparative analysis against a number of the most prolific PqsR antagonists recently developed. The bioluminescent PQS-based biosensor for the detection of PA was engineered to respond to the presence of exogenous PQS that forms a complex with the regulatory PqsR protein, ultimately stimulating the expression of a luxCDABE-fused pqsA promoter. The biosensor was subsequently inserted in a non-pathogenic E. coli recipient by means of chromosomal integration, devoid of the sdiA LuxR homolog that could potentially interfere with the recognition of PqsR. A silent reporter was observed when in E. coli, but further assessments to its genetic integrity did not reveal any single nucleotide polymorphisms (SNP). In addition, after further testings to its activity in different established PA mutants, devoid of genes that constituted the bioreporting system or to it directly associated, a fully functional bioreporter was confirmed. Finally, a few possible explanations as to what might be in the origin of a defective bioreporter in E. coli are discussed. Lastly, a new LESA-MS protocol based on the surface sampling of dried bacterial extracts, envisaging its potentialities as a rapid and cheap screening method for detection of AQs, was designed and optimised. Even though the method has been widely used in a variety of research scenarios, this is the first time LESA-MS is applied as a screening methodology in the context of bacterial extract screening. Overall, the optimisation process showed that LESA-MS is an approach with numerous potentialities and immediate advantages, where one emphasises sampling simplicity, fast delivering of results, sensitivity to AQs at the nanomolar level (especially for C7-PQS and the precursor HHQ). But simultaneously, this methodology also revealed limitations inherent to its setting up that constrain an effective screening. The most emphatic ones being the volatility of the preparations to intra-sampling variability, and to a certain degree, an unexpected insensitivity to important QS N-acyl homoserine lactones (AHLs), namely C4-HSL and 3-oxo-C12-HSL. Nevertheless, such limitations do not present themselves as an insurmountable barrier, and based on results from available studies making use of the LESA-MS a number of possibilities to work around these are also presented

Use of transgenic GFP reporter strains of the nematode Caenorhabditis elegans to investigate the patterns of stress responses induced by pesticides and by organic extracts from agricultural soils

As a free-living nematode, C. elegans is exposed to various pesticides used in agriculture, as well as to persistent organic residues which may contaminate the soil for long periods. Following on from our previous study of metal effects on 24 GFP-reporter strains representing four different stress-response pathways in C. elegans (Anbalagan et al. 2012), we now present parallel data on the responses of these same strains to several commonly used pesticides. Some of these, like dichlorvos, induced multiple stress genes in a concentration-dependent manner. Unusually, endosulfan induced only one gene (cyp-34A9) to very high levels (8-10-fold) even at the lowest test concentration, with a clear plateau at higher doses. Other pesticides, like diuron, did not alter reporter gene expression detectably even at the highest test concentration attainable, while others (such as glyphosate) did so only at very high concentrations. We have also used five responsive GFP reporters to investigate the toxicity of soil pore water from two agricultural sites in south-east Spain, designated P74 (used for cauliflower production, but significantly metal contaminated) and P73 (used for growing lettuce, but with only background levels of metals). Both soil pore water samples induced all five test genes to varying extents, yet artificial mixtures containing all major metals present had essentially no effect on these same transgenes. Soluble organic contaminants present in the pore water were extracted with acetone and dichloromethane, then after evaporation of the solvents, the organic residues were redissolved in ultrapure water to reconstitute the soluble organic components of the original soil pore water. These organic extracts induced transgene expression at similar or higher levels than the original pore water. Addition of the corresponding metal mixtures had either no effect, or reduced transgene expression towards the levels seen with soil pore water only. We conclude that the main toxicants present in these soil pore water samples are organic rather than metallic in nature. Organic extracts from a control standard soil (Lufa 2.2) had negligible effects on expression of these genes, and similarly several pesticides had little effect on the expression of a constitutive myo-3::GFP transgene. Both the P73 and P74 sites have been treated regularly with (undisclosed) pesticides, as permitted under EU regulations, though other (e.g. industrial) organic residues may also be present

Hsp90 orchestrates transcriptional regulation by Hsf1 and cell wall remodelling by MAPK signalling during thermal adaptation in a pathogenic yeast

Acknowledgments We thank Rebecca Shapiro for creating CaLC1819, CaLC1855 and CaLC1875, Gillian Milne for help with EM, Aaron Mitchell for generously providing the transposon insertion mutant library, Jesus Pla for generously providing the hog1 hst7 mutant, and Cathy Collins for technical assistance.Peer reviewedPublisher PD

Lessons Learned Developing a Diagnostic Tool for HIV-Associated Dementia Feasible to Implement in Resource-Limited Settings: Pilot Testing in Kenya

Objective: To conduct a preliminary evaluation of the utility and reliability of a diagnostic tool for HIV-associated dementia (HAD) for use by primary health care workers (HCW) which would be feasible to implement in resource-limited settings. Background: In resource-limited settings, HAD is an indication for anti-retroviral therapy regardless of CD4 T-cell count. Anti-retroviral therapy, the treatment for HAD, is now increasingly available in resource-limited settings. Nonetheless, HAD remains under-diagnosed likely because of limited clinical expertise and availability of diagnostic tests. Thus, a simple diagnostic tool which is practical to implement in resource-limited settings is an urgent need. Methods: A convenience sample of 30 HIV-infected outpatients was enrolled in Western Kenya. We assessed the sensitivity and specificity of a diagnostic tool for HAD as administered by a primary HCW. This was compared to an expert clinical assessment which included examination by a physician, neuropsychological testing, and in selected cases, brain imaging. Agreement between HCW and an expert examiner on certain tool components was measured using Kappa statistic. Results: The sample was 57 % male, mean age was 38.6 years, mean CD4 T-cell count was 323 cells/mL, and 54 % had less than a secondary school education. Six (20%) of the subjects were diagnosed with HAD by expert clinical assessment. The diagnostic tool was 63 % sensitive and 67 % specific for HAD. Agreement between HCW and expert examiners was poor for many individual items of the diagnostic tool (K =.03–.65). This diagnostic tool had moderate sensitivity and specificity fo

Transgenic nematodes as biosensors for metal stress in soil pore water samples

Caenorhabditis elegans strains carrying stress-reporter green fluorescent protein transgenes were used to explore patterns of response to metals. Multiple stress pathways were induced at high doses by most metals tested, including members of the heat shock, oxidative stress, metallothionein (mtl) and xenobiotic response gene families. A mathematical model (to be published separately) of the gene regulatory circuit controlling mtl production predicted that chemically similar divalent metals (classic inducers) should show additive effects on mtl gene induction, whereas chemically dissimilar metals should show interference. These predictions were verified experimentally; thus cadmium and mercury showed additive effects, whereas ferric iron (a weak inducer) significantly reduced the effect of mercury. We applied a similar battery of tests to diluted samples of soil pore water extracted centrifugally after mixing 20% w/w ultrapure water with air-dried soil from an abandoned lead/zinc mine in the Murcia region of Spain. In addition, metal contents of both soil and soil pore water were determined by ICP-MS, and simplified mixtures of soluble metal salts were tested at equivalent final concentrations. The effects of extracted soil pore water (after tenfold dilution) were closely mimicked by mixtures of its principal component ions, and even by the single most prevalent contaminant (zinc) alone, though other metals modulated its effects both positively and negatively. In general, mixtures containing similar (divalent) metal ions exhibited mainly additive effects, whereas admixture of dissimilar (e.g. trivalent) ions often resulted in interference, reducing overall levels of stress-gene induction. These findings were also consistent with model predictions

LASER PHYSICS LETTERS

Abstract: Raman spectroscopy offers a powerful alternative analytical method for the detection and identification of lipids/oil in biological samples, such as algae and fish. Recent research in the authors' groups, and experimental data only very recently published by us and a few other groups suggest that Raman spectroscopy can be exploited in instances where fast and accurate determination of the iodine value (associated with the degree of lipid unsaturation) is required. Here the current status of Raman spectroscopy applications on algae is reviewed, and particular attention is given to the efforts of identifying and selecting oil-rich algal strains for the potential mass production of commercial biofuels and for utilization in the food industry. Normalized intensity, a.u

PACS: 32.30.-r, 32.60.+i, 32.70

Abstract: We have measured light shifts, also known as AC Stark shifts, as a function of laser intensity in cold Rubidium atoms by observing sub-natural linewidth gain and loss features in the transmission spectrum of a weak probe beam passing through the atomic sample. The observed energy-level shifts for atoms in a magneto-optical trap (MOT) are found to be consistently higher than that obtained in optical molasses (i.e., when the magnetic field gradient in the MOT is turned off). Using a simple model of a multilevel Rubidium atom interacting with pump and probe beams, we have calculated the theoretical light shift as a function of intensity. A comparison of these calculated values with the light shift data obtained for molasses reveals good agreement between experiment and theory. Further, our model elucidates the role of the Zeeman shifts arising from the magnetic field gradient in the observed probe transmission spectrum for the MOT. A qualitative plot of the transmission spectrum of a probe beam through a fictitious sample of cold J = 1 → J = 2 atoms showing probe absorption at the sum of the pump frequency ω pump and δ , where δ is the difference of the light shifts between the |J = 1,mJ = 0 and the |J = 1,mJ = ± 1 ground state Zeeman sublevels. Probe gain is depicted at ω pump -δ . Se