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

A Novel OxyR Sensor and Regulator of Hydrogen Peroxide Stress with One Cysteine Residue in Deinococcus radiodurans

In bacteria, OxyR is a peroxide sensor and transcription regulator, which can sense the presence of reactive oxygen species and induce antioxidant system. When the cells are exposed to H2O2, OxyR protein is activated via the formation of a disulfide bond between the two conserved cysteine residues (C199 and C208). In Deinococcus radiodurans, a previously unreported special characteristic of DrOxyR (DR0615) is found with only one conserved cysteine. dr0615 gene mutant is hypersensitive to H2O2, but only a little to ionizing radiation. Site-directed mutagenesis and subsequent in vivo functional analyses revealed that the conserved cysteine (C210) is necessary for sensing H2O2, but its mutation did not alter the binding characteristics of OxyR on DNA. Under oxidant stress, DrOxyR is oxidized to sulfenic acid form, which can be reduced by reducing reagents. In addition, quantitative real-time PCR and global transcription profile results showed that OxyR is not only a transcriptional activator (e.g., katE, drb0125), but also a transcriptional repressor (e.g., dps, mntH). Because OxyR regulates Mn and Fe ion transporter genes, Mn/Fe ion ratio is changed in dr0615 mutant, suggesting that the genes involved in Mn/Fe ion homeostasis, and the genes involved in antioxidant mechanism are highly cooperative under extremely oxidant stress. In conclusion, these findings expand the OxyR family, which could be divided into two classes: typical 2-Cys OxyR and 1-Cys OxyR

Impaired dispersion correlates with increased persistence as determined using a chronic murine pneumonia infection model.

<p>CD1 mice were inoculated intratracheal through oral lavage using feeding needle with 1.2×10⁶ CFU of <i>Pseudomonas</i> strains. Lungs were harvested 14 days post-inoculation and CFU was determined. A total of 10 mice were used in per study. (A) Bacterial burden in the lung. Values presented are average, min, max, mean lung CFU/ml. (B) Fold change in lung CFU/ml 14 days post-infection compared to initial inoculum. Error bars indicate standard deviation. The values were tested by means of a Fisher test. **, significantly different from PAO1, P-value <0.001.</p

BdlA, DipA and Induced Dispersion Contribute to Acute Virulence and Chronic Persistence of <i>Pseudomonas aeruginosa</i>

<div><p>The human pathogen <i>Pseudomonas aeruginosa</i> 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 <i>P. aeruginosa</i> 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 Δ<i>bdlA</i> biofilms were characterized by a lack of many of the degradative activities. Expression of genes contributing to the virulence of <i>P. aeruginosa</i> 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 Δ<i>bdlA</i> and Δ<i>dipA</i> biofilms compared to wild-type planktonic cells. Despite this, <i>bdlA</i> and <i>dipA</i> inactivation, resulting in an inability to disperse <i>in vitro</i>, correlated with reduced pathogenicity and competitiveness in cross-phylum acute virulence models. In contrast, <i>bdlA</i> inactivation rendered <i>P. aeruginosa</i> more persistent upon chronic colonization of the murine lung, overall indicating that dispersion may contribute to both acute and chronic infections.</p></div

Identification of proteins present in supernatants.

<p>Identification of proteins present in supernatants.</p

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

Analysis of proteins present in supernatants of <i>P.</i> aeruginosa PAO1 and <i>ΔbdlA</i> biofilms.

<p>A total of 10 µg supernatant protein obtained from <i>P. aeruginosa</i> PAO1 and <i>ΔbdlA</i> biofilms was loaded per lane. Protein bands indicated by a letter and arrow were selected and subsequently identified by LC-MS/MS (see also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004168#ppat-1004168-t001" target="_blank">Table 1</a>). Experiments were repeated in triplicate and a representative SDS-gel image is shown. Molecular masses are indicated on the right (in kDa).</p

The non-dispersing Δ<i>bdlA</i> mutant and complemented Δ<i>bdlA</i> mutants impaired in biofilm dispersion are avirulent.

<p>(A) Death of <i>Arabidopsis thaliana</i> 7 days post infection with <i>P. aeruginosa</i> PAO1, the isogenic Δ<i>bdlA</i> mutant and <i>ΔbdlA</i> mutants complemented with <i>bdlA</i>, a truncated BdlA variant (NoPAS-<i>bdlA</i>) and BdlA variants harboring alanine substitutions in various amino acids. #, indicates complemented Δ<i>bdlA</i> strains impaired in nutrient-induced dispersion, see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004168#ppat.1004168-Petrova6" target="_blank">[58]</a>. All other complemented Δ<i>bdlA</i> strains were not impaired in nutrient-induced dispersion. (B) Death of <i>Arabidopsis thaliana</i> 7 days post-infection with <i>P. aeruginosa</i> PAO1 and selected isogenic mutants. Bars indicate average and median plant death rates while vertical lines indicate the highest and lowest plant death rates observed. (C) Death of <i>Arabidopsis thaliana</i> 7 days post infection with <i>P. aeruginosa</i> PA14, and the isogenic Δ<i>dipA</i> and <i>ΔrbdA</i> mutants. Control plants inoculated with ½ MS salts alone showed no symptoms over the course of the experiments. Experiments were carried out in triplicate using 8 plants per strain per replicate. *, significantly different from PAO1, P-value <0.05.</p

Dispersion of <i>P. aeruginosa</i> PAO1 biofilms correlates with increased release of proteins into the supernatant.

<p>Supernatants were obtained from (A) <i>P. aeruginosa</i> PAO1 and (B) Δ<i>bdlA</i> grown planktonically to exponential and stationary phase, as well as from biofilms and cells dispersed from the biofilm in response to exposure to glutamate (dispersed cells). Dispersed cells were obtained following dispersion in response to glutamate and SNP, which was used as a source of nitric oxide. Likewise, planktonic cells grown to exponential and stationary phase were exposed for 20 min to glutamate or SNP. Planktonic cells not treated with glutamate or nitric oxide are referred to as “control”. Experiments were carried out in triplicate. Error bars indicate standard deviation. The protein concentration of supernatants was determined using the same number of cells (1x10⁹ CFU/ml) regardless of growth conditions. n.d., not determined.</p