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

Pseudomonas aeruginosa is a significant nosocomial pathogen and is associated with lung infections in cystic fibrosis (CF). Once established, P. aeruginosa 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 P. aeruginosa senses, responds to, and protects itself against HOCl and HOSCN and the contribution of such responses to its success as a CF pathogen. To investigate the P. aeruginosa response to these oxidants, we screened 707 transposon mutants, with mutations in regulatory genes, for altered growth following HOCl exposure. We identified regulators of antibiotic resistance, methionine biosynthesis, catabolite repression, and PA14_07340, the homologue of the Escherichia coli HOCl-sensor RclR (30% identical), which are required for protection against HOCl. We have shown that RclR (PA14_07340) protects specifically against HOCl and HOSCN stress and responds to both oxidants by upregulating the expression of a putative peroxiredoxin, rclX (PA14_07355). Transcriptional analysis revealed that while there was specificity in the response to HOCl (231 genes upregulated) and HOSCN (105 genes upregulated), there was considerable overlap, with 74 genes upregulated by both oxidants. These included genes encoding the type 3 secretion system, sulfur and taurine transport, and the MexEF-OprN efflux pump. RclR coordinates part of the response to both oxidants, including upregulation of pyocyanin biosynthesis genes, and, in the presence of HOSCN, downregulation of chaperone genes. These data indicate that the P. aeruginosa response to HOCl and HOSCN is multifaceted, with RclR playing an essential role.IMPORTANCE The bacterial pathogen Pseudomonas aeruginosa causes devastating infections in immunocompromised hosts, including chronic lung infections in cystic fibrosis patients. To combat infection, the host's immune system produces the antimicrobial oxidants hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN). Little is known about how P. aeruginosa responds to and survives attack from these oxidants. To address this, we carried out two approaches: a mutant screen and transcriptional study. We identified the P. aeruginosa transcriptional regulator, RclR, which responds specifically to HOCl and HOSCN stress and is essential for protection against both oxidants. We uncovered a link between the P. aeruginosa transcriptional response to these oxidants and physiological processes associated with pathogenicity, including antibiotic resistance and the type 3 secretion system.

Cyclic di-adenosine monophosphate (c-di-AMP) is required for osmotic regulation in Staphylococcus aureus 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 Staphylococcus aureus However, the exact role of this molecule has not been identified. dacA, the S. aureus 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 S. aureus dacA mutant can be generated in chemically defined medium. Consistent with previous findings, this mutant had a severe growth defect when cultured in rich medium. Using this growth defect in rich medium, we selected for suppressor strains with improved growth to identify c-di-AMP-requiring pathways. Mutations bypassing the essentiality of dacA were identified in alsT and opuD, encoding a predicted amino acid and osmolyte transporter, the latter of which we show here to be the main glycine betaine-uptake system in S. aureus. Inactivation of these transporters likely prevents the excessive osmolyte and amino acid accumulation in the cell, providing further evidence for a key role of c-di-AMP in osmotic regulation. Suppressor mutations were also obtained in hepS, hemB, ctaA, and qoxB, coding proteins required for respiration. Furthermore, we show that dacA is dispensable for growth in anaerobic conditions. Together, these findings reveal an essential role for the c-di-AMP signaling network in aerobic, but not anaerobic, respiration in S. aureus.

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. Better detection of P. aeruginosa infection using biomarkers may enable more successful eradication before chronic infection is established. In chronic infection P. aeruginosa adapts to avoid immune clearance and resist antibiotics via efflux pumps, ß-lactamase expression, reduced porins and switching to a biofilm lifestyle. The optimal treatment strategies for P. aeruginosa infection are still being established, and new antibiotic formulations such as liposomal amikacin, fosfomycin in combination with tobramycin and inhaled levofloxacin are being explored. Novel agents such as the alginate oligosaccharide OligoG, cysteamine, bacteriophage, nitric oxide, garlic oil and gallium may be useful as anti-pseudomonal strategies, and immunotherapy to prevent infection may have a role in the future. New treatments that target the primary defect in cystic fibrosis, recently licensed for use, have been associated with a fall in P. aeruginosa infection prevalence. Understanding the mechanisms for this could add further strategies for treating P. aeruginosa in future.

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. Chronic infection is associated with accelerated disease progression and increased mortality. Extensive research has revealed complex mechanisms by which P. aeruginosa adapts to and persists within the CF airway. Yet knowledge gaps remain, and prevention and treatment strategies are limited by the lack of sensitive detection methods and by a narrow armoury of antibiotics. Further developments in this field are urgently needed in order to improve morbidity and mortality in people with CF. Here, we summarize current knowledge of pathophysiological mechanisms underlying P. aeruginosa infection in CF. Established treatments are discussed, and an overview is offered of novel detection methods and therapeutic strategies in development.

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. aeruginosa, and compared the wild-type strains with deletion mutants for crc. A complex metabolite consumption hierarchy was observed, which was broadly similar between the two species, although with some important differences, for example in sugar utilization. The order of metabolite utilization changed upon inactivation of the crc gene, but even in the Crc-null strains some compounds were completely consumed before late metabolites were taken up. This suggests the presence of additional regulatory elements that determine the time and order of consumption of compounds. Unexpectedly, the loss of Crc led both species to excrete acetate and pyruvate as a result of unbalanced growth during exponential phase, compounds that were later consumed in stationary phase. This loss of carbon during growth helps to explain the contribution of the Crc/Hfq regulatory system to evolutionary fitness of pseudomonads.

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. Potassium cyanide decreased ciliary beat frequency in healthy nasal brushings (n = 6) after 60 min (150 µM: 47% fall, p<0.0012; 75 µM: 32% fall, p<0.0001). Samples from cystic fibrosis patients (n = 3) showed similar results (150 µM: 55% fall, p = 0.001). Ciliary beat frequency inhibition was not due to loss of cell viability and was reversible. The inhibitory mechanism was independent of ATP levels. KCN also significantly inhibited ciliary beat frequency in ALI cultures, albeit to a lesser extent. Ciliary beat frequency measurements on ALI cultures treated with culture supernatants from P. aeruginosa mutants defective in virulence factor production implicated cyanide as a key component inhibiting the ciliary beat frequency. If cyanide production similarly impairs mucocilliary clearance in vivo, it could explain the link with increased disease severity observed in cystic fibrosis patients with detectable cyanide in their airway.

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. Tandem affinity purification using the His- and Strep-tags is well-suited to the isolation of protein complexes with a high purity and no detectable background. We have validated this approach using the well-described pyruvate dehydrogenase complex.

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. Here, we describe time-resolved metabolic footprinting (TReF), a technique which employs nuclear magnetic resonance spectroscopy and nonlinear curve fitting to understand and visualize metabolite utilization of P. aeruginosa.

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.

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. A phenotypic and gene expression characterisation of the stationary phase physiology of mucA22 mutants demonstrated complex and subtle changes in virulence factor production, including cyanide and pyocyanin, that results in their down-regulation upon entry into stationary phase but, (and in contrast to wildtype strains) continued production in prolonged stationary phase. These findings may have consequences for chronic infection if mucoid P. aeruginosa were to continue to make virulence factors under non-growing conditions during infection. These changes resulted in part from a severe down-regulation of both AHL-and AQ (PQS)-dependent quorum sensing systems. In trans expression of the cAMP-dependent transcription factor Vfr restored both quorum sensing defects and virulence factor production in early stationary phase. Our findings have implications for understanding the evolution of P. aeruginosa during CF lung infection and it demonstrates that mucA22 mutation provides a second mechanism, in addition to the commonly occurring lasR mutations, of down-regulating quorum sensing during chronic infection this may provide a selection pressure for the mucoid switch in the CF lung.

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. While the individual functions of these genes are unknown, with one exception (i.e. a ferredoxin-dependent reductase), a highly inducible six-gene cluster (PA4129-PA4134) was found to be crucial for protection of P. aeruginosa from external HCN intoxication. A double mutant deleted for PA4129-PA4134 and cioAB (encoding cyanide-insensitive oxidase) did not grow with 100 µM KCN, whereas the corresponding single mutants were essentially unaffected, suggesting a synergistic action of the PA4129-PA4134 gene products and cyanide-insensitive oxidase.

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. E.g. for two-component system mutants, the cognate response regulator and sensor kinase genes clustered tightly together. Some strains had metabolic phenotypes (metabotypes) that could be related to the known gene function. E.g. pyruvate dehydrogenase mutants accumulated large amounts of pyruvate in the medium. In other cases, the metabolic phenotypes were not easily interpretable. The rpoN mutant, which lacks the alternative σ factor RpoN (σ(54)), accumulated high levels of gluconate in the medium. In addition, endometabolome profiling of intracellular metabolites identified a number of systemic metabolic changes. We linked this to indirect regulation of the catabolite repression protein Crc via the non-coding RNA crcZ and found that a crcZ (but not crc) mutant also shared the high-gluconate phenotype. We profiled an additional set of relevant metabolic enzymes and transporters, including Crc targets, and showed that the Crc-regulated edd mutant (gluconate-6-phosphate dehydratase) had similar gluconate levels as the rpoN mutant. Finally, a set of clinical isolates showed patient- and random amplification of polymorphic DNA (RAPD) type-specific differences in gluconate production, which were associated significantly with resistance across four antibiotics (tobramycin, ciprofloxacin, aztreonam, and imipenem), indicating that this has potential clinical relevance.

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., acetate was produced only in unfiltered ASM.

The dormancy regulator DosR controls ribosome stability in hypoxic mycobacteria.

It is thought that during latent infection, Mycobacterium tuberculosis bacilli are retained within granulomas in a low-oxygen environment. The dormancy survival (Dos) regulon, regulated by the response regulator DosR, appears to be essential for hypoxic survival in M. tuberculosis, but it is not known how the regulon promotes survival. Here we report that mycobacteria, in contrast to enteric bacteria, do not form higher-order structures (e.g. ribosomal dimers) upon entry into stasis. Instead, ribosomes are stabilized in the associated form (70S). Using a strategy incorporating microfluidic, proteomic, and ribosomal profiling techniques to elucidate the fate of mycobacterial ribosomes during hypoxic stasis, we show that the dormancy regulator DosR is required for optimal ribosome stabilization. We present evidence that the majority of this effect is mediated by the DosR-regulated protein MSMEG_3935 (a S30AE domain protein), which is associated with the ribosome under hypoxic conditions. A Δ3935 mutant phenocopies the ΔdosR mutant during hypoxia, and complementation of ΔdosR with the MSMEG_3935 gene leads to complete recovery of dosR mutant phenotypes during hypoxia. We suggest that this protein is named ribosome-associated factor under hypoxia (RafH) and that it is the major factor responsible for DosR-mediated hypoxic survival in mycobacteria.

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). The speed and simplicity of this approach could accelerate the study of many different aspects of ribosomal biology.

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. However, apart from the mucoid phenotype little is known about the overall metabolic and physiological changes caused by mucA mutation. We investigated the pleiotropic metabolic effects of this mutation using time-resolved metabolic footprinting (extracellular metabolomics), and found changes in the levels of various metabolites associated with osmotic tolerance, including glycine-betaine, trehalose and glutamate. Physiological experiments confirmed that the isogenic mucA22 mutant is less resistant to osmotic stress than the parental PA01 wild-type strain, but only in the stationary phase of growth. Quantitative comparison of the endometabolome of the cells showed differences in the accumulation of osmoprotective metabolites by the wild-type and mucA22 mutant strains, suggesting a switch in osmo-protectant preference from glycine-betaine to trehalose.

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. Strikingly, while HS therapy does not alter the prevalence of P. aeruginosa in the CF lung it does decrease the frequency of episodes of acute, severe illness known as infective exacerbations among CF patients. In this article, we propose a hypothesis whereby the positive clinical effects of HS treatment are explained by the osmotic sensitivity of the mucoid sub-population of P. aeruginosa in the CF lung leading to selection against this group in favor of the osmotically resistant non-mucoid variants.

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. In addition, metabolite excretion showed some complex patterns. Fitting nonlinear equations (four-parameter sigmoids) to individual metabolite data allowed us to model these changes for metabolite uptake and visualize them by back-projecting the curve-fit parameters onto the original growth curves. These "uptake window" plots clearly demonstrated strain differences, with the uptake of some compounds being reversed in order between different strains. Comparison of an undefined rich medium with a defined complex medium designed to mimic cystic fibrosis sputum showed many differences, both qualitative and quantitative, with a greater proportion of excreted to utilized metabolites in the defined medium. Extending the strain comparison to a more closely related set of isolates showed that it was possible to discriminate two species of the Burkholderia cepacia complex based on uptake dynamics alone. We believe time-resolved metabolic footprinting could be a valuable tool for many questions in bacteriology, including isolate comparisons, phenotyping deletion mutants, and as a functional complement to taxonomic classifications.

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. Pseudomonas aeruginosa is another important CF pathogen. It is able to synthesise hydrogen cyanide (HCN), a potent inhibitor of cellular respiration. We have recently shown that HCN production by P. aeruginosa may have a role in CF pathogenesis. This paper describes an investigation of the ability of bacteria of the Bcc to make HCN. RESULTS: The genome of Burkholderia cenocepacia has 3 putative HCN synthase encoding (hcnABC) gene clusters. B. cenocepacia and all 9 species of the Bcc complex tested were able to make cyanide at comparable levels to P. aeruginosa, but only when grown surface attached as colonies or during biofilm growth on glass beads. In contrast to P. aeruginosa and other cyanogenic bacteria, cyanide was not detected during planktonic growth of Bcc strains. CONCLUSION: All species in the Bcc are cyanogenic when grown as surface attached colonies or as biofilms.