An Overview of Infections in Cystic Fibrosis Airways and the Role of Environmental Conditions on Pseudomonas aeruginosa Biofilm Formation and Viability

In this chapter, the authors review a major complication associated with cystic fibrosis (CF), problematic bacterial infections of the lungs. Infection by organisms such as Staphylococcus aureus, Burkholderia cepacia complex, and Pseudomonas aeruginosa (a major player in CF related infections) results in complications due to increased inflammation and production of virulence factors produced by the bacteria. In addition to these more canonical organisms associated with CF infection, emergingbacterial species have been found in the CF, including anaerobes that have only within the past 5-10 years have been reported to exist in the lungs. P. aeruginosa has long been a cause of devastating infections, and is often seen as the“hallmark”organism associated with the disease. The authors describe the P. aeruginosa infection, including its conversion to a mucoid phenotype, as well as its ability to utilize the thicker airway surface layer associated with CF to grow in “mode two biofilms.” Finally, the authors discuss treatments for bacterial infections, and some of the new advances that offerhope for treatment of CF symptoms and infections by multi-drug resistant organisms. Among these new treatments is the application of acidified nitrite, a non-antibiotic treatment that has been found to be effective at killing nonmucoid and mucoid variants of P. aeruginosa

BdlA, DipA and Induced Dispersion Contribute to Acute Virulence and Chronic Persistence of Pseudomonas aeruginosa

The human pathogen Pseudomonas aeruginosa is capable of causing both acute and chronic infections. Differences in virulence are attributable to the mode of growth: bacteria growing planktonically cause acute infections, while bacteria growing in matrix-enclosed aggregates known as biofilms are associated with chronic, persistent infections. While the contribution of the planktonic and biofilm modes of growth to virulence is now widely accepted, little is known about the role of dispersion in virulence, the active process by which biofilm bacteria switch back to the planktonic mode of growth. Here, we demonstrate that P. aeruginosa dispersed cells display a virulence phenotype distinct from those of planktonic and biofilm cells. While the highest activity of cytotoxic and degradative enzymes capable of breaking down polymeric matrix components was detected in supernatants of planktonic cells, the enzymatic activity of dispersed cell supernatants was similar to that of biofilm supernatants. Supernatants of non-dispersing Delta bdlA biofilms were characterized by a lack of many of the degradative activities. Expression of genes contributing to the virulence of P. aeruginosa was nearly 30-fold reduced in biofilm cells relative to planktonic cells. Gene expression analysis indicated dispersed cells, while dispersing from a biofilm and returning to the single cell lifestyle, to be distinct from both biofilm and planktonic cells, with virulence transcript levels being reduced up to 150-fold compared to planktonic cells. In contrast, virulence gene transcript levels were significantly increased in non-dispersing Delta bdlA and Delta dipA biofilms compared to wild-type planktonic cells. Despite this, bdlA and dipA inactivation, resulting in an inability to disperse in vitro, correlated with reduced pathogenicity and competitiveness in cross-phylum acute virulence models. In contrast, bdlA inactivation rendered P. aeruginosa more persistent upon chronic colonization of the murine lung, overall indicating that dispersion may contribute to both acute and chronic infections

The Anti-Sigma Factor MucA of Pseudomonas aeruginosa: Dramatic Differences of a mucA22 vs. a ΔmucA Mutant in Anaerobic Acidified Nitrite Sensitivity of Planktonic and Biofilm Bacteria in vitro and During Chronic Murine Lung Infection

Mucoid mucA22 Pseudomonas aeruginosa (PA) is an opportunistic lung pathogen of cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) patients that is highly sensitive to acidified nitrite (A-NO2-). In this study, we first screened PA mutant strains for sensitivity or resistance to 20 mM A-NO2- under anaerobic conditions that represent the chronic stages of the aforementioned diseases. Mutants found to be sensitive to A-NO2- included PA0964 (pmpR, PQS biosynthesis), PA4455 (probable ABC transporter permease), katA (major catalase, KatA) and rhlR (quorum sensing regulator). In contrast, mutants lacking PA0450 (a putative phosphate transporter) and PA1505 (moaA2) were A-NO2- resistant. However, we were puzzled when we discovered that mucA22 mutant bacteria, a frequently isolated mucA allele in CF and to a lesser extent COPD, were more sensitive to A-NO2- than a truncated ΔmucA deletion (Δ157–194) mutant in planktonic and biofilm culture, as well as during a chronic murine lung infection. Subsequent transcriptional profiling of anaerobic, A-NO2--treated bacteria revealed restoration of near wild-type transcript levels of protective NO2- and nitric oxide (NO) reductase (nirS and norCB, respectively) in the ΔmucA mutant in contrast to extremely low levels in the A-NO2--sensitive mucA22 mutant. Proteins that were S-nitrosylated by NO derived from A-NO2- reduction in the sensitive mucA22 strain were those involved in anaerobic respiration (NirQ, NirS), pyruvate fermentation (UspK), global gene regulation (Vfr), the TCA cycle (succinate dehydrogenase, SdhB) and several double mutants were even more sensitive to A-NO2-. Bioinformatic-based data point to future studies designed to elucidate potential cellular binding partners for MucA and MucA22. Given that A-NO2- is a potentially viable treatment strategy to combat PA and other infections, this study offers novel developments as to how clinicians might better treat problematic PA infections in COPD and CF airway diseases

Global regulation of gene expression by OxyR in an important human opportunistic pathogen

Most bacteria control oxidative stress through the H2O2-responsive transactivator OxyR, a member of the LTTR family (LysR Type Transcriptional Regulators), which activates the expression of defensive genes such as those encoding catalases, alkyl hydroperoxide reductases and superoxide dismutases. In the human opportunistic pathogen Pseudomonas aeruginosa, OxyR positively regulates expression of the oxidative stress response genes katA, katB, ahpB and ahpCF. To identify additional targets of OxyR in P. aeruginosa PAO1, we performed chromatin immunoprecipitation in combination with whole genome tiling array analyses (ChIP-chip). We detected 56 genes including all the previously identified defensive genes and a battery of novel direct targets of OxyR. Electrophoretic mobility shift assays (EMSAs) for selected newly identified targets indicated that ∼70% of those were bound by purified oxidized OxyR and their regulation was confirmed by quantitative real-time polymerase chain reaction. Furthermore, a thioredoxin system was identified to enzymatically reduce OxyR under oxidative stress. Functional classification analysis showed that OxyR controls a core regulon of oxidative stress defensive genes, and other genes involved in regulation of iron homeostasis (pvdS), quorum-sensing (rsaL), protein synthesis (rpsL) and oxidative phosphorylation (cyoA and snr1). Collectively, our results indicate that OxyR is involved in oxidative stress defense and regulates other aspects of cellular metabolism as well

Novel Organic Hydroperoxide-Sensing and Responding Mechanisms for OhrR, a Major Bacterial Sensor and Regulator of Organic Hydroperoxide Stress

Xanthomonas campestris pv. phaseoli OhrR belongs to a major family of multiple-cysteine-containing bacterial organic hydroperoxide sensors and transcription repressors. Site-directed mutagenesis and subsequent in vivo functional analyses revealed that changing any cysteine residue to serine did not alter the ability of OhrR to bind to the P1 ohrR-ohr promoter but drastically affected the organic hydroperoxide-sensing and response mechanisms of the protein. Xanthomonas OhrR requires two cysteine residues, C22 and C127, to sense and respond to organic hydroperoxides. Analysis of the free thiol groups in wild-type and mutant OhrRs under reducing and oxidizing conditions indicates that C22 is the organic hydroperoxide-sensing residue. Exposure to organic hydroperoxides led to the formation of an unstable OhrR-C22 sulfenic acid intermediate that could be trapped by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and detected by UV-visible spectral analysis in an oxidized C127S-C131S mutant OhrR. In wild-type OhrR, the cysteine sulfenic acid intermediate rapidly reacts with the thiol group of C127, forming a disulfide bond. The high-performance liquid chromatography-mass spectrometry analysis of tryptic fragments of alkylated, oxidized OhrR and nonreducing polyacrylamide gel electrophoresis analyses confirmed the formation of reversible intersubunit disulfide bonds between C22 and C127. Oxidation of OhrR led to cross-linking of two OhrR monomers, resulting in the inactivation of its repressor function. Evidence presented here provides insight into a new organic hydroperoxide-sensing and response mechanism for OhrRs of the multiple-cysteine family, the primary bacterial transcription regulator of the organic hydroperoxide stress response

Sodium Nitrite-Mediated Killing of the Major Cystic Fibrosis Pathogens Pseudomonas aeruginosa, Staphylococcus aureus, and Burkholderia cepacia under Anaerobic Planktonic and Biofilm Conditions▿

A hallmark of airways in patients with cystic fibrosis (CF) is highly refractory, chronic infections by several opportunistic bacterial pathogens. A recent study demonstrated that acidified sodium nitrite (A-NO2−) killed the highly refractory mucoid form of Pseudomonas aeruginosa, a pathogen that significantly compromises lung function in CF patients (S. S. Yoon et al., J. Clin. Invest. 116:436-446, 2006). Therefore, the microbicidal activity of A-NO2− (pH 6.5) against the following three major CF pathogens was assessed: P. aeruginosa (a mucoid, mucA22 mutant and a sequenced nonmucoid strain, PAO1), Staphylococcus aureus USA300 (methicillin resistant), and Burkholderia cepacia, a notoriously antibiotic-resistant organism. Under planktonic, anaerobic conditions, growth of all strains except for P. aeruginosa PAO1 was inhibited by 7.24 mM (512 μg ml−1 NO2−). B. cepacia was particularly sensitive to low concentrations of A-NO2− (1.81 mM) under planktonic conditions. In antibiotic-resistant communities known as biofilms, which are reminiscent of end-stage CF airway disease, A-NO2− killed mucoid P. aeruginosa, S. aureus, and B. cepacia; 1 to 2 logs of cells were killed after a 2-day incubation with a single dose of ∼15 mM A-NO2−. Animal toxicology and phase I human trials indicate that these bactericidal levels of A-NO2− can be easily attained by aerosolization. Thus, in summary, we demonstrate that A-NO2− is very effective at killing these important CF pathogens and could be effective in other infectious settings, particularly under anaerobic conditions where bacterial defenses against the reduction product of A-NO2−, nitric oxide (NO), are dramatically reduced

The Peptidoglycan-Associated Lipoprotein OprL Helps Protect a Pseudomonas aeruginosa Mutant Devoid of the Transactivator OxyR from Hydrogen Peroxide-Mediated Killing during Planktonic and Biofilm Culture ▿ †

OxyR controls H2O2-dependent gene expression in Pseudomonas aeruginosa. Without OxyR, diluted (<107/ml) organisms are easily killed by micromolar H2O2. The goal of this study was to define proteins that contribute to oxyR mutant survival in the presence of H2O2. We identified proteins in an oxyR mutant that were oxidized by using 2,4-dinitrophenylhydrazine for protein carbonyl detection, followed by identification using a two-dimensional gel/matrix-assisted laser desorption ionization-time of flight approach. Among these was the peptidoglycan-associated lipoprotein, OprL. A double oxyR oprL mutant was constructed and was found to be more sensitive to H2O2 than the oxyR mutant. Provision of the OxyR-regulated alkyl hydroperoxide reductase, AhpCF, but not AhpB or the catalase, KatB, helped protect this strain against H2O2. Given the sensitivity of oxyR oprL bacteria to planktonic H2O2, we next tested the hypothesis that the biofilm mode of growth might protect such organisms from H2O2-mediated killing. Surprisingly, biofilm-grown oxyR oprL mutants, which (in contrast to planktonic cells) possessed no differences in catalase activity compared to the oxyR mutant, were sensitive to killing by as little as 0.5 mM H2O2. Transmission electron microscopy studies revealed that the integrity of both cytoplasmic and outer membranes of oxyR and oxyR oprL mutants were compromised. These studies suggest that sensitivity to the important physiological oxidant H2O2 in the exquisitely sensitive oxyR mutant bacteria is based not only upon the presence and location of OxyR-controlled antioxidant enzymes such as AhpCF but also on structural reinforcement by the peptidoglycan-associated lipoprotein OprL, especially during growth in biofilms

Detection of degradative activity in the extracellular proteome of <i>P. aeruginosa</i> PAO1 is growth mode dependent with <i>P. aeruginosa ΔbdlA</i> impaired in dispersion exhibiting lower degradative activity.

<p>Cytotoxic and degradative activities were determined using 10 µg of supernatant protein in 100 µl of sterile water, followed by measuring the zone of clearance 18 hr post-inoculation of the sterile protein solution into the wells of the respective agar plates. Degradative activity was determined using supernatants obtained from <i>P. aeruginosa</i> grown planktonically to exponential (A) and stationary phase (B). Supernatants of planktonic cells not treated with glutamate or nitric oxide are referred to as “control”. Additionally, supernatants of planktonic cells grown to exponential and stationary phase were exposed for 30 min to glutamate or SNP were used. (C–F) Degradative activities were furthermore determined in supernatants obtained from biofilms, and biofilms post-induction of dispersion with glutamate (remaining biofilm). Dispersed cells were obtained following dispersion in response to glutamate and SNP, which was used as a source of nitric oxide. (C) Proteolytic activity was detected using milk agar plates in supernatants obtained from biofilms, biofilms post-induction of dispersion, and dispersed cells. (D) Lipid hydrolysis was determined using tributyrin containing agar plates. (E) Hemolytic activity was detected using blood agar plates while (F) Psl degradation was detected on agar plates containing Psl extracted from a <i>P. aeruginosa</i> strain overexpressing Psl. Psl degradation was visualized as a zone of clearing following 24 hr incubation and staining the agar plate with iodine. Experiments were carried out at least in triplicate. Error bars indicate standard deviation.</p

The OxyR-regulated <i>phnW</i> gene encoding 2-aminoethylphosphonate:pyruvate aminotransferase helps protect <i>Pseudomonas aeruginosa</i> from <i>tert</i>-butyl hydroperoxide

<div><p>The LysR member of bacterial transactivators, OxyR, governs transcription of genes involved in the response to H₂O₂ and organic (alkyl) hydroperoxides (AHP) in the Gram-negative pathogen, <i>Pseudomonas aeruginosa</i>. We have previously shown that organisms lacking OxyR are rapidly killed by <2 or 500 mM H₂O₂ in planktonic and biofilm bacteria, respectively. In this study, we first employed a bioinformatic approach to elucidate the potential regulatory breadth of OxyR by scanning the entire <i>P</i>. <i>aeruginosa</i> PAO1 genome for canonical OxyR promoter recognition sequences (ATAG-N₇-CTAT-N₇-ATAG-N₇-CTAT). Of >100 potential OxyR-controlled genes, 40 were strategically selected that were <u><b><i>not</i></b></u> predicted to be involved in the direct response to oxidative stress (e.g., catalase, peroxidase, etc.) and screened such genes by RT-PCR analysis for potentially positive or negative control by OxyR. Differences were found in 7 of 40 genes when comparing an <i>oxyR</i> mutant vs. PAO1 expression that was confirmed by ß-galactosidase reporter assays. Among these, <i>phnW</i>, encoding 2-aminoethylphosphonate:pyruvate aminotransferase, exhibited reduced expression in the <i>oxyR</i> mutant compared to wild-type bacteria. Electrophoretic mobility shift assays indicated binding of OxyR to the <i>phnW</i> promoter and DNase I footprinting analysis also revealed the sequences to which OxyR bound. Interestingly, a <i>phnW</i> mutant was more susceptible to <i>t</i>-butyl-hydroperoxide (<i>t</i>-BOOH) treatment than wild-type bacteria. Although we were unable to define the direct mechanism underlying this phenomenon, we believe that this may be due to a reduced efficiency for this strain to degrade <i>t</i>-BOOH relative to wild-type organisms because of modulation of AHP gene transcription in the <i>phnW</i> mutant.</p></div