Response of Pseudomonas aeruginosa to the Innate Immune System-Derived Oxidants Hypochlorous Acid and Hypothiocyanous Acid.

is a significant nosocomial pathogen and is associated with lung infections in cystic fibrosis (CF). Once established, infections persist and are rarely eradicated despite host immune cells producing antimicrobial oxidants, including hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN). There is limited knowledge as to how senses, responds to, and protects itself against HOCl and HOSCN and the contribution of such responses to its success as a CF pathogen.

Cyclic di-adenosine monophosphate (c-di-AMP) is required for osmotic regulation in but dispensable for viability in anaerobic conditions.

Cyclic di-adenosine monophosphate (c-di-AMP) is a recently discovered signaling molecule important for the survival of Firmicutes, a large bacterial group that includes notable pathogens such as However, the exact role of this molecule has not been identified. , the gene encoding the diadenylate cyclase enzyme required for c-di-AMP production, cannot be deleted when bacterial cells are grown in rich medium, indicating that c-di-AMP is required for growth in this condition. Here, we report that an mutant can be generated in chemically defined medium.

Current and future therapies for Pseudomonas aeruginosa infection in patients with cystic fibrosis.

Pseudomonas aeruginosa opportunistically infects the airways of patients with cystic fibrosis and causes significant morbidity and mortality. Initial infection can often be eradicated though requires prompt detection and adequate treatment. Intermittent and then chronic infection occurs in the majority of patients.

Pseudomonas aeruginosa infection in cystic fibrosis: pathophysiological mechanisms and therapeutic approaches.

Pseudomonas aeruginosa is a remarkably versatile environmental bacterium with an extraordinary capacity to infect the cystic fibrosis (CF) lung. Infection with P. aeruginosa occurs early, and although eradication can be achieved following early detection, chronic infection occurs in over 60% of adults with CF.

Influence of the Crc regulator on the hierarchical use of carbon sources from a complete medium in Pseudomonas.

The Crc protein, together with the Hfq protein, participates in catabolite repression in pseudomonads, helping to coordinate metabolism. Little is known about how Crc affects the hierarchy of metabolite assimilation from complex mixtures. Using proton Nuclear Magnetic Resonance (NMR) spectroscopy, we carried out comprehensive metabolite profiling of culture supernatants (metabolic footprinting) over the course of growth of both Pseudomonas putida and P.

Cyanide levels found in infected cystic fibrosis sputum inhibit airway ciliary function.

We have previously reported cyanide at concentrations of up to 150 μM in the sputum of cystic fibrosis patients infected with Pseudomonas aeruginosa and a negative correlation with lung function. Our aim was to investigate possible mechanisms for this association, focusing on the effect of pathophysiologically relevant cyanide levels on human respiratory cell function. Ciliary beat frequency measurements were performed on nasal brushings and nasal air-liquid interface (ALI) cultures obtained from healthy volunteers and cystic fibrosis patients.

An in vivo crosslinking system for identifying mycobacterial protein-protein interactions.

The analysis of protein-protein interactions in Mycobacterium tuberculosis has the potential to shed light on the functions of the large number of predicted open-reading frames annotated as conserved hypothetical proteins. We have developed a formaldehyde crosslinking system to detect in vivo interactions in mycobacteria. Our Gateway-adapted vector system uses three promoter strengths, including constitutive and regulatable versions, for the expression of target proteins with either an N- or C-terminal His-Strep-Strep tag.

The mucoid switch in Pseudomonas aeruginosa represses quorum sensing systems and leads to complex changes to stationary phase virulence factor regulation.

The opportunistic pathogen Pseudomonas aeruginosa chronically infects the airways of Cystic Fibrosis (CF) patients during which it adapts and undergoes clonal expansion within the lung. It commonly acquires inactivating mutations of the anti-sigma factor MucA leading to a mucoid phenotype, caused by excessive production of the extracellular polysaccharide alginate that is associated with a decline in lung function. Alginate production is believed to be the key benefit of mucA mutations to the bacterium in the CF lung.

Cyanide measurements in bacterial culture and sputum.

Cyanide is produced by a few bacterial species, including Pseudomonas aeruginosa, and it has a role in the opportunistic infections of this bacterium including in cystic fibrosis lung infections. We describe two methods for determining cyanide in culture and patient sputum samples. One uses an ion-selective electrode to provide a convenient, rapid method of cyanide quantitation in culture or sputum, and the second is a semiquantitative method using Feigl-Anger paper that is useful for screening large numbers of bacterial strains for cyanide production.

Metabolic footprinting: extracellular metabolomic analysis.

Uptake and excretion of nutrients is an integral part of a cell's physiology. Using analytical chemistry techniques, metabolite uptake and excretion from the culture medium can be quantified. As cellular metabolism changes throughout growth, additional information is available if transient and growth phase-dependent changes are monitored.

A novel cyanide-inducible gene cluster helps protect Pseudomonas aeruginosa from cyanide.

Pseudomonas aeruginosa produces the toxic secondary metabolite hydrogen cyanide (HCN) at high cell population densities and low aeration. Here, we investigated the impact of HCN as a signal in cell-cell communication by comparing the transcriptome of the wild-type strain PAO1 to that of an HCN-negative mutant under cyanogenic conditions. HCN repressed four genes and induced 12 genes.

Metabolite profiling to characterize disease-related bacteria: gluconate excretion by Pseudomonas aeruginosa mutants and clinical isolates from cystic fibrosis patients.

Metabolic footprinting of supernatants has been proposed as a tool for assigning gene function. We used NMR spectroscopy to measure the exometabolome of 86 single-gene transposon insertion mutant strains (mutants from central carbon metabolism and regulatory mutants) of the opportunistic pathogen Pseudomonas aeruginosa, grown on a medium designed to represent the nutritional content of cystic fibrosis sputum. Functionally related genes had similar metabolic profiles.

Direct assessment of metabolite utilization by Pseudomonas aeruginosa during growth on artificial sputum medium.

We grew Pseudomonas aeruginosa in LB and artificial sputum medium (ASM) (filtered and unfiltered) and quantified metabolite utilization and excretion by nuclear magnetic resonance (NMR) spectroscopy (metabolic footprinting or extracellular metabolomics). Utilization rates were similar between media, but there were differences in excretion-e.g.

Isolation of bacterial ribosomes with monolith chromatography.

We report the development of a rapid chromatographic method for the isolation of bacterial ribosomes from crude cell lysates in less than ten minutes. Our separation is based on the use of strong anion exchange monolithic columns. Using a simple stepwise elution program we were able to purify ribosomes whose composition is comparable to those isolated by sucrose gradient ultracentrifugation, as confirmed by quantitative proteomic analysis (iTRAQ).

Metabolic profiling of Pseudomonas aeruginosa demonstrates that the anti-sigma factor MucA modulates osmotic stress tolerance.

Metabolic footprinting has shown enormous potential as a phenotyping tool and we are interested in applying it to understand the physiology of the opportunistic pathogen Pseudomonas aeruginosa during its chronic infection of the lungs of cystic fibrosis patients. The selection pressures of surviving in the CF lung environment lead to genetic adaptations of the bacterium. A common adaptation is mutation of the mucA gene, resulting in a loss-of-function mutation to the anti-sigma factor MucA, which leads to a mucoid phenotype as a consequence of the overproduction of the extracellular polysaccharide alginate.

Hypertonic Saline Therapy in Cystic Fibrosis: Do Population Shifts Caused by the Osmotic Sensitivity of Infecting Bacteria Explain the Effectiveness of this Treatment?

Cystic fibrosis (CF) is caused by a defect in the CF transmembrane regulator that leads to depletion and dehydration of the airway surface liquid (ASL) of the lung epithelium, providing an environment that can be infected by bacteria leading to increased morbidity and mortality. Pseudomonas aeruginosa chronically infects more than 80% of CF patients and one hallmark of infection is the emergence of a mucoid phenotype associated with a worsening prognosis and more rapid decline in lung function. Hypertonic saline (HS) is a clinically proven treatment that improves mucociliary clearance through partial rehydration of the ASL of the lung.

Time-resolved metabolic footprinting for nonlinear modeling of bacterial substrate utilization.

Untargeted profiling of small-molecule metabolites from microbial culture supernatants (metabolic footprinting) has great potential as a phenotyping tool. We used time-resolved metabolic footprinting to compare one Escherichia coli and three Pseudomonas aeruginosa strains growing on complex media and show that considering metabolite changes over the whole course of growth provides much more information than analyses based on data from a single time point. Most strikingly, there was pronounced selectivity in metabolite uptake, even when the bacteria were growing apparently exponentially, with certain groups of metabolites not taken up until others had been entirely depleted from the medium.

Bacteria of the Burkholderia cepacia complex are cyanogenic under biofilm and colonial growth conditions.

Background: The Burkholderia cepacia complex (Bcc) is a collection of nine genotypically distinct but phenotypically similar species. They show wide ecological diversity and include species that are used for promoting plant growth and bio-control as well species that are opportunistic pathogens of vulnerable patients. Over recent years the Bcc have emerged as problematic pathogens of the CF lung.

The coordinate regulation of multiple terminal oxidases by the Pseudomonas putida ANR global regulator.

Pseudomonas putida KT2440 contains a branched aerobic respiratory chain with multiple terminal oxidases. Their relative proportion varies according to environmental conditions. The role of the oxygen-responsive ANR global regulator on expression of these terminal oxidases was analysed.

Oxygen, cyanide and energy generation in the cystic fibrosis pathogen Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a gram-negative, rod-shaped bacterium that belongs to the gamma-proteobacteria. This clinically challenging, opportunistic pathogen occupies a wide range of niches from an almost ubiquitous environmental presence to causing infections in a wide range of animals and plants. P.

Investigation of the physiological relationship between the cyanide-insensitive oxidase and cyanide production in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen which demonstrates considerable respiratory versatility, possessing up to five terminal oxidases. One oxidase, the cyanide-insensitive oxidase (CIO), has been previously shown to be resistant to the potent respiratory inhibitor cyanide, a toxin that is synthesized by this bacterium. This study investigated the physiological relationship between hydrogen cyanide production and the CIO.

Defects in a quinol oxidase lead to loss of KatC catalase activity in Pseudomonas aeruginosa: KatC activity is temperature dependent and it requires an intact disulphide bond formation system.

Mutation or overexpression of the cyanide-insensitive terminal oxidase (CIO) of Pseudomonas aeruginosa leads to temperature-sensitivity, multiple antibiotic sensitivity, and abnormal cell division and failure to produce a temperature-inducible catalase [G.R. Tavankar, D.

Global analysis of proteins synthesized by Mycobacterium smegmatis provides direct evidence for physiological heterogeneity in stationary-phase cultures.

We have characterized the induction kinetics of approximately 1,700 proteins during entry into and survival in carbon-starved stationary phase by Mycobacterium smegmatis. Strikingly, among the patterns of expression observed were a group of proteins that were expressed in exponential-phase cultures and severely repressed in 48-h stationary-phase cultures (Spr or stationary-phase-repressed proteins) but were synthesized again at high levels in > or =128-day stationary-phase cultures (Spr(128) proteins). A number of Spr(128) proteins were identified, and they included the heat shock protein DnaK, the tricarboxylic acid cycle enzyme succinyl coenzyme A synthase, a FixA-like flavoprotein, a single-stranded DNA binding protein, and elongation factor Tu (EF-Tu).