Investigation of global regulators influencing styrene metabolism and bioplastic synthesis in Pseudomonas putida CA-3

The genetics and biochemistry involved in the biodegradation of styrene and the production of polyhydroxyalkanoates in Pseudomonas putida CA-3 have been well characterised to date. Knowledge of the role played by global regulators in controlling these pathways currently represents a critical knowledge gap in this area. Here we report on our efforts to identify such regulators using mini-Tn5 transposon mutagenesis of the P. putida CA-3 genome. The library generated was subjected to phenotypic screening to identify mutants exhibiting a reduced sensitivity to the effects of carbon catabolite repression of aromatic pathway activity. Our efforts identified a clpX disrupted mutant which exhibited wild-type levels of growth on styrene but significantly reduced growth on phenylacetic acid. RT-PCR analysis of key PACoA catabolon genes necessary for phenylacetic acid metabolism, and SDS-PAGE protein profile analyses suggest that no direct alteration of PACoA pathway transcriptional or translational activity was involved. The influence of global regulators affecting the accumulation of PHAs in P. putida CA-3 was also studied. Phenotypic screening of the mini-Tn5 library revealed a gacS sensor kinase gene disruption resulting in the loss of PHA accumulation capacity in P. putida CA-3. Subsequent SDS-PAGE protein analyses of the wild type and gacS mutant strains identified post-transcriptional control of phaC1 synthase as a key point of control of PHA synthesis in P. putida CA-3. Disruption of the gacS gene in another PHA accumulating organism, P. putida S12, also demonstrated a reduction of PHA accumulation capacity. PHA accumulation was observed to be disrupted in the CA-3 gacS mutant under phosphorus limited growth conditions. Over-expression studies in both wild type CA-3 and gacS mutant demonstrated that rsmY over-expression in gacS disrupted P. putida CA-3 is insufficient to restore PHA accumulation in the cell however in wild type cells, over-expression of rsmY results in an altered PHA monomer compositions.

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.

Microbial inactivation of Pseudomonas putida and Pichia pastoris using gene silencing.

Antisense deoxyoligonucleotide (ASO) gene silencing was investigated as a potential disinfection tool for industrial and drinking water treatment application. ASOs bind with their reverse complementary mRNA transcripts thereby blocking protein translation. While ASO silencing has mainly been studied in medicine, it may be useful for modulating gene expression and inactivating microorganisms in environmental applications. In this proof of concept work, gene targets were sh ble (zeocin resistance) and todE (catechol-2,3-dioxygenase) in Pichia pastoris and npt (kanamycin resistance) in Pseudomonas putida. A maximum 0.5-fold decrease in P. pastoris cell numbers was obtained following a 120 min incubation with single-stranded DNA (ssDNA) concentrations ranging from 0.2 to 200 nM as compared to the no ssDNA control. In P. putida, a maximum 5.2-fold decrease was obtained after 90 min with 400 nM ssDNA. While the silencing efficiencies varied for the 25 targets tested, these results suggest that protein activity as well as microbial growth can be altered using ASO gene silencing-based tools. If successful, this technology has the potential to eliminate some of the environmental and health issues associated with the use of strong chemical biocides. However, prior to its dissemination, more research is needed to increase silencing efficiency and develop effective delivery methods.

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.

Elective versus symptomatic antibiotic treatment in cystic fibrosis patients with chronic Pseudomonas infection of the lungs

BACKGROUND:
A previous retrospective study suggested that a policy of regular anti-pseudomonal antibiotic treatment improved pulmonary function and increased survival in patients with cystic fibrosis chronically infected with Pseudomonas species. The results of a prospective multicentre study to compare the effects on pulmonary function and mortality of three monthly elective anti-pseudomonal antibiotic treatment with conventional symptomatic treatment are reported.

METHODS:
Sixty patients with cystic fibrosis, chronically infected with P aeruginosa, were randomised to the two treatment arms (elective or symptomatic) and followed clinically at yearly reviews. The major end points were changes in forced expiratory volume in one second (FEV(1)) and forced vital capacity (FVC). Survival was a secondary end point.

RESULTS:
Patients in the symptomatic group received a mean of three antibiotic treatments each year and those in the elective group received four antibiotic treatments during each year of the study. No significant differences in FEV(1) and FVC were found between the two groups after three years. There was a statistically non-significant higher rate of deaths in the elective group (n = 4), three of which were associated with B cepacia infection, compared with the symptomatic group (n = 0).

CONCLUSIONS:
This study did not demonstrate an advantage of a policy of elective antibiotic treatment over symptomatic treatment in patients with cystic fibrosis chronically infected with Pseudomonas species.

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.