Manipulation of Pseudomonas aeruginosa alginate pathway by varying the level of biosynthetic enzymes and growth temperature

J.H. LEITÃO AND i. SÁ-CORREIA. 1993. The manipulation of the alginate pathway in two Pseudomonas aeruginosa mucoid variants was attempted at growth temperatures within the range 20C-40C. This was carried out by increasing the level of either phosphomannose isomerase (PMI) and GDP-mannose pyrophosphorylase (GMP) or GDP-mannose dehydrogenase (GMD) encoded by algA or algD respectively, present in recombinant plasmids derived from the controlled expression vector pMMB24. The specific growth rate of cells expressing either algA or algD genes from recombinant plasmids was lower than that of cells harbouring the cloning vector only. Stimulation of alginate synthesis was observed when the expression of the alginate genes was low, in the absence of isopropyl-β-D-thiogalactopyranoside (IPTG) induction. The further increase of the level of alginate enzymes in induced cells, without the simultaneous increase of other limiting steps, had no positive effect on the strictly regulated alginate pathway. Temperature profiles for alginate synthesis were modified reflecting changes in rate limiting steps. Limitations on the polymerization ability and the competition between cell growth and alginate synthesis were possibly involved in the modification of the temperature profiles for alginate production, or in the decrease of the molecular weight of polymers produced by recombinants under conditions that led to highly active alginate synthesis. The acetyl content of alginates produced by the recombinants was higher than that of the biopolymer controls, possibly due to the higher acetyl-CoA availability in slower growing cells.     

Molecular modeling and redesign of alginate lyase from Pseudomonas aeruginosa for accelerating CRPA biofilm degradation

Administration of an efficient alginate lyase (AlgL) or AlgL mutant may be a promising therapeutic strategy for treatment of cystic fibrosis patients with Pseudomonas aeruginosa infections. Nevertheless, the catalytic activity of wild‐type AlgL is not sufficiently high. It is highly desired to design and discover an AlgL mutant with significantly improved catalytic efficiency against alginate substrates. For the purpose of identifying an AlgL mutant with significantly improved catalytic activity, in this study, we first constructed and validated a structural model of AlgL interacting with substrate, providing a better understanding of the interactions between AlgL and its substrate. Based on the modeling insights, further enzyme redesign and experimental testing led to discovery of AlgL mutants, including the K197D/K321A mutant, with significantly improved catalytic activities against alginate and acetylated alginate in ciprofloxacin‐resistant P. aeruginosa (CRPA) biofilms. Further anti‐biofilm activity assays have confirmed that the K197D/K321A mutant with piperacillin/tazobactam is indeed effective in degrading the CRPA biofilms. Co‐administration of the potent mutant AlgL and an antibiotic (such as a nebulizer) could be effective for therapeutic treatment of CRPA‐infected patients with cystic fibrosis. Proteins 2016; 84:1875–1887. © 2016 Wiley Periodicals, Inc.     

Somatostatin regulates brain amyloid beta peptide Abeta42 through modulation of proteolytic degradation

Expression of somatostatin in the brain declines during aging in various mammals including apes and humans. A prominent decrease in this neuropeptide also represents a pathological characteristic of Alzheimer disease. Using in vitro and in vivo paradigms, we show that somatostatin regulates the metabolism of amyloid beta peptide (Abeta), the primary pathogenic agent of Alzheimer disease, in the brain through modulating proteolytic degradation catalyzed by neprilysin. Among various effector candidates, only somatostatin upregulated neprilysin activity in primary cortical neurons. A genetic deficiency of somatostatin altered hippocampal neprilysin activity and localization, and increased the quantity of a hydrophobic 42-mer form of Abeta, Abeta(42), in a manner similar to presenilin gene mutations that cause familial Alzheimer disease. These results indicate that the aging-induced downregulation of somatostatin expression may be a trigger for Abeta accumulation leading to late-onset sporadic Alzheimer disease, and suggest that somatostatin receptors may be pharmacological-target candidates for prevention and treatment of Alzheimer disease.     

Exopolysaccharide production in biofilms: substratum activation of alginate gene expression by Pseudomonas aeruginosa.

Reporter gene technology was employed to detect the activity of an alginate promoter of Pseudomonas aeruginosa when the organism was grown as a biofilm on a Teflon mesh substratum and as planktonic cells in liquid medium. Alginate biosynthetic activity was determined with a mucoid cell line derived from a cystic fibrosis isolate and containing an alginate algC promoter fused to a lacZ reporter gene. Reporter activity was demonstrated with chromogenic and fluorogenic substrates for beta-galactosidase. Expression of algC was shown to be upregulated in biofilm cells compared with planktonic cells in liquid medium. Gene up-expression correlated with alginate biosynthesis as measured by Fourier transform infrared spectroscopy, uronic acid accumulation, and alginate-specific enzyme-linked immunosorbent assay. The algC promoter was shown to have maximum activity in planktonic cultures during the late lag and early log phases of the cell growth cycle. During a time course experiment, biofilm algC activity exceeded planktonic activity except during the period immediately following inoculation into fresh medium. In continuous-culture experiments, conversion of lacZ substrate was demonstrated microscopically in individual cells by epifluorescence microscopy.     

Effect of Pseudomonas aeruginosa crude proteolytic fraction on antibacterial activity of Galleria mellonella haemolymph

The antibacterial activity of immune haemolymph Galleria mellonella directed against Escherichia coli D31 was destroyed by Pseudomonas aeruginosa crude proteolytic fraction. This was demonstrated by diffusion well assay and acid gel electrophoresis and subsequent bioautography. On the contrary, lysozyme activity appeared to be insensitive to extracellular proteases of P. aeruginosa when activity was determined using the bioautography method. In addition, no change in lysozyme protein level was observed by immunoblotting with specific antibodies directed against G. mellonella lysozyme, which confirmed that lysozyme was not degraded by the crude proteolytic fraction of P. aeruginosa. However, a significant decrease of lysozyme activity in naive and immune haemolymph exposed to the action of P. aeruginosa proteins determined by using diffusion well assay was observed. Mechanisms of the observed inhibition require further studies.     

Pseudomonas aeruginosa alkaline protease blocks complement activation via the classical and lectin pathways

The complement system rapidly detects and kills Gram-negative bacteria and supports bacterial killing by phagocytes. However, bacterial pathogens exploit several strategies to evade detection by the complement system. The alkaline protease (AprA) of Pseudomonas aeruginosa has been associated with bacterial virulence and is known to interfere with complement-mediated lysis of erythrocytes, but its exact role in bacterial complement escape is unknown. In this study, we analyzed how AprA interferes with complement activation and whether it could block complement-dependent neutrophil functions. We found that AprA potently blocked phagocytosis and killing of Pseudomonas by human neutrophils. Furthermore, AprA inhibited opsonization of bacteria with C3b and the formation of the chemotactic agent C5a. AprA specifically blocked C3b deposition via the classical and lectin pathways, whereas the alternative pathway was not affected. Serum degradation assays revealed that AprA degrades both human C1s and C2. However, repletion assays demonstrated that the mechanism of action for complement inhibition is cleavage of C2. In summary, we showed that P. aeruginosa AprA interferes with classical and lectin pathway-mediated complement activation via cleavage of C2.     

Alginate lyase (AlgL) activity is required for alginate biosynthesis in Pseudomonas aeruginosa

To determine whether AlgL's lyase activity is required for alginate production in Pseudomonas aeruginosa, an algLdelta::Gm(r) mutant (FRD-MA7) was created. algL complementation of FRD-MA7 restored alginate production, but algL constructs containing mutations inactivating lyase activity did not, demonstrating that the enzymatic activity of AlgL is required for alginate production.     

Lipopolysaccharide-induced Notch signaling activation through JNK-dependent pathway regulates inflammatory response

Background  

Notch and TLR pathways were found to act cooperatively to activate Notch target genes and to increase the production of TLR-induced cytokines in macrophages. However, the mechanism of LPS-induced Notch activation and its role in sepsis still remains unclear.     

Composition of alginate synthesized during the growth cycle of Pseudomonas aeruginosa

A mucoid P. aeruginosa isolated from the sputum of a cystic fibrosis patient was grown in batch culture on a complex medium. During the growth cycle the amount of alginate produced was estimated and its composition was determined by proton magnetic resonance (1H-n.m.r) spectroscopy. Exopolysaccharide production occurred mainly during the exponential phase of growth. The alginate samples isolated varied little in composition and were characterized by being highly acetylated, high mannuronate (0.83-0.93 mole fraction) polymers. Guluronate was present only within heteropolymeric regions of the polysaccharides which all displayed a complete absence of polyguluronate. Ca2+ ion supplementation of the medium was not observed to increase the levels of guluronate in the alginates produced.     

Real-time PCR used to measure stress-induced changes in the expression of the genes of the alginate pathway of Pseudomonas aeruginosa

AIMS: To measure the concentration of mRNAs transcribed from four genes involved in alginate production using real-time PCR. METHODS AND RESULTS: The mRNA concentrations in cells grown in normal and stress conditions were compared. A difference in the expression of algD, the key gene leading to overproduction of alginate, was detected between alginate-producing and non-alginate-producing strains grown under normal conditions. After growth on 3% ethanol (known to stimulate alginate production), but not after heat-shock, an increase in algD mRNA levels and a corresponding decrease in mucB (a regulatory gene) mRNA levels were detected in all strains. CONCLUSION: The quantitative results suggest that the mucB gene may have a role in recognition of stress conditions, and that having a disrupted mucA gene does not always result in a mucoid phenotype. SIGNIFICANCE AND IMPACT OF THE STUDY: Real-time PCR can be used to quantify mRNA and is a convenient method of analysing bacterial gene expression.     

Emergence of nylon oligomer degradation enzymes in Pseudomonas aeruginosa PAO through experimental evolution.

Through selective cultivation with 6-aminohexanoate linear dimer, a by-product of nylon-6 manufacture, as the sole source of carbon and nitrogen, Pseudomonas aeruginosa PAO, which initially has no enzyme activity to degrade this xenobiotic compound, was successfully expanded in its metabolic ability. Two new enzyme activities, 6-aminohexanoate cyclic dimer hydrolase and 6-aminohexanoate dimer hydrolase, were detected in the adapted strains.