The interaction of recombinant Pseudomonas aeruginosa exotoxin A with DNA and mimic biomembrane investigated by surface plasmon resonance and electrochemical methods

Based on the multidomain structure of Pseudomonas aeruginosa exotoxin A, a fusion protein termed rPEA has been constructed, which is expected to serve as a gene carrier in vitro. The expression and purification of rPEA are described. The basal properties of rPEA as a gene carrier are evaluated by investigating its interaction with plasmid DNA and mimic biomembrane by surface plasmon resonance (SPR) and electrochemical methods. rPEA is proved to be able to bind with plasmid DNA with high affinity. It can also interact with lipid membrane and increase permeability of the membrane, so the probe molecules can easily reach the gold surface and exhibit the electrochemical response.

Characterising novel anti-biofilm targets for the treatment of chronic Pseudomonas aeruginosa infection in the cystic fibrosis lung

The global rise in antibiotic resistance is a significant problem facing healthcare professionals. In particular within the cystic fibrosis (CF) lung, bacteria can establish chronic infection and resistance to a wide array of antibiotic therapies. One of the principle pathogens associated with chronic infection in the CF lung is Pseudomonas aeruginosa. P. aeruginosa can establish chronic infection in the CF lung partly through the use of the biofilm mode of growth. This biofilm mode of growth offers a considerable degree of protection from a wide variety of challenges such as the host immune system or antibiotic therapy. The threat posed by the emergence of chronic pathogens is prompting the development of next generation antimicrobials. The biofilm mode of growth is often central to the establishment of chronic infection and the development of antibiotic resistance. Thus, targeting biofilm formation has emerged as one of the principle strategies for the development of next generation antimicrobials. In this thesis two separate approaches were used to identify potential anti - biofilm targets. The first strategy focused on the identification of novel genes with a role in a biofilm formation. High throughput screening identified almost 300 genes which had a role in biofilm formation. A number of these genes were characterised at a phenotypic and a molecular level. The second strategy focused on the identification of compounds capable of inhibiting biofilm formation. A collection of marine sponge isolated bacteria were screened for the ability to inhibit the central pathway regulating biofilm formation, quorum sensing. A number of distinct isolates were identified that had quorum sensing inhibition activity from which, a Pseudomonas isolate was selected for further characterisation. A specific compound capable of inhibiting quorum sensing was identified using chemical analytical technologies in the supernatant of this marine isolate.

Using sediment flocculation to reduce the impacts of Chesapeake Bay Microcystis aeruginosa harmful algal blooms

Gemstone Team BREATHE (Bay Revitalization Efforts Against the Hypoxic Environment)

Stress-induced outer membrane vesicle production by Pseudomonas aeruginosa.

As an opportunistic Gram-negative pathogen, Pseudomonas aeruginosa must be able to adapt and survive changes and stressors in its environment during the course of infection. To aid survival in the hostile host environment, P. aeruginosa has evolved defense mechanisms, including the production of an exopolysaccharide capsule and the secretion of a myriad of degradative proteases and lipases. The production of outer membrane-derived vesicles (OMVs) serves as a secretion mechanism for virulence factors as well as a general bacterial response to envelope-acting stressors. This study investigated the effect of sublethal physiological stressors on OMV production by P. aeruginosa and whether the Pseudomonas quinolone signal (PQS) and the MucD periplasmic protease are critical mechanistic factors in this response. Exposure to some environmental stressors was determined to increase the level of OMV production as well as the activity of AlgU, the sigma factor that controls MucD expression. Overexpression of AlgU was shown to be sufficient to induce OMV production; however, stress-induced OMV production was not dependent on activation of AlgU, since stress caused increased vesiculation in strains lacking algU. We further determined that MucD levels were not an indicator of OMV production under acute stress, and PQS was not required for OMV production under stress or unstressed conditions. Finally, an investigation of the response of P. aeruginosa to oxidative stress revealed that peroxide-induced OMV production requires the presence of B-band but not A-band lipopolysaccharide. Together, these results demonstrate that distinct mechanisms exist for stress-induced OMV production in P. aeruginosa.

Pseudomonas aeruginosa vesicles associate with and are internalized by human lung epithelial cells.

BACKGROUND: Pseudomonas aeruginosa is the major pathogen associated with chronic and ultimately fatal lung infections in patients with cystic fibrosis (CF). To investigate how P. aeruginosa-derived vesicles may contribute to lung disease, we explored their ability to associate with human lung cells. RESULTS: Purified vesicles associated with lung cells and were internalized in a time- and dose-dependent manner. Vesicles from a CF isolate exhibited a 3- to 4-fold greater association with lung cells than vesicles from the lab strain PAO1. Vesicle internalization was temperature-dependent and was inhibited by hypertonic sucrose and cyclodextrins. Surface-bound vesicles rarely colocalized with clathrin. Internalized vesicles colocalized with the endoplasmic reticulum (ER) marker, TRAPalpha, as well as with ER-localized pools of cholera toxin and transferrin. CF isolates of P. aeruginosa abundantly secrete PaAP (PA2939), an aminopeptidase that associates with the surface of vesicles. Vesicles from a PaAP knockout strain exhibited a 40% decrease in cell association. Likewise, vesicles from PAO1 overexpressing PaAP displayed a significant increase in cell association. CONCLUSION: These data reveal that PaAP promotes the association of vesicles with lung cells. Taken together, these results suggest that P. aeruginosa vesicles can interact with and be internalized by lung epithelial cells and contribute to the inflammatory response during infection.

Development of a rapid colorimetric time-kill assay for determining the in vitro activity of ceftazidime and tobramycin in combination against Pseudomonas aeruginosa

It is standard clinical practice to use a combination of two or more antimicrobial agents to treat an infection caused by Pseudonionas aeruginosa. The antibiotic combinations are usually selected empirically with methods to determine the antimicrobial effect of the combination such as the time-kill assay rarely used as they are time-consuming and labour intensive to perforin. Here, we report a modified time-kill assay, based on the reduction of the tetrazolium salt, 2,3-bis[2-methyloxy-4-nitro-5-sulfopheny1]-2H-tetrazolium-5-carboxanilide (XTT), that allows simple, inexpensive and more rapid determination of the in vitro activity of antibiotic combinations against P aeruginosa. The assay was used to determine the in vitro activity of ceftazidime and tobramycin in combination against P. aertiginosa isolates from cystic fibrosis patients and the results obtained compared with those from conventional viable count time-kill assays. There was good agreement in interpretation of results obtained by the XTT and conventional viable count assays, with similar growth curves apparent and the most effective concentration combinations determined by both methods identical for all isolates tested. The XTT assay clearly indicated whether an antibiotic combination had a synergistic, indifferent or antagonistic effect and could, therefore, provide a useful method for rapidly determining the activity of a large number of antibiotic combinations against clinical isolates. (C) 2004 Elsevier B.V. All rights reserved.