Blue light treatment of Pseudomonas aeruginosa: Strong bactericidal activity, synergism with antibiotics and inactivation of virulence factors.

Pseudomonas aeruginosa is among the most common pathogens responsible for both acute and chronic infections of high incidence and severity. Additionally, P. aeruginosa resistance to conventional antimicrobials has increased rapidly over the past decade. Therefore, it is crucial to explore new therapeutic options, particularly options that specifically target the pathogenic mechanisms of this microbe. The ability of a pathogenic bacterium to cause disease is dependent upon the production of agents termed 'virulence factors', and approaches to mitigate these agents have gained increasing attention as new antibacterial strategies. Although blue light irradiation is a promising alternative approach, only limited and preliminary studies have described its effect on virulence factors. The current study aimed to investigate the effects of lethal and sub-lethal doses of blue light treatment (BLT) on P. aeruginosa virulence factors. We analyzed the inhibitory effects of blue light irradiation on the production/activity of several virulence factors. Lethal BLT inhibited the activity of pyocyanin, staphylolysin, pseudolysin and other proteases, but sub-lethal BLT did not affect the production/expression of proteases, phospholipases, and flagella- or type IV pili-associated motility. Moreover, a eukaryotic cytotoxicity test confirmed the decreased toxicity of blue light-treated extracellular P. aeruginosa fractions. Finally, the increased antimicrobial susceptibility of P. aeruginosa treated with sequential doses of sub-lethal BLT was demonstrated with a checkerboard test. Thus, this work provides evidence-based proof of the susceptibility of drug-resistant P. aeruginosa to BLT-mediated killing, accompanied by virulence factor reduction, and describes the synergy between antibiotics and sub-lethal BLT.

How to avoid premature decay of your macromolecular crystal: a quick soak for long life.

Radiation damage to biological samples is currently one of the major limiting factors in macromolecular X-ray crystallography, since it severely and irreversibly affects the quality of the data that can be obtained from a diffraction experiment. However, radiation damage can effectively be reduced by utilizing the electron and radical scavenging potential of certain small-molecule compounds. We propose an approach to protect macromolecular crystals prior to data collection by quick soaking with scavengers. This, in favorable cases, can more than double crystal lifetime in the X-ray beam. The approach has the potential to yield diffraction data of superior quality and hence to increase the amount of high-quality diffraction data and of structural information attainable from a single crystal.

Pathophysiology of photoaging of human skin: focus on neutrophils.

UV-induced skin damage is the result of a complex cascade of events. Many studies have focused on the skin effects induced by UV-B or UV-A separately. Recently a UV-source that emits UV-B and UV-A together in a ratio comparable to daily sunlight has been introduced: i.e. solar simulated radiation (SSR). By exposing human skin type I-III to erythematogenic doses of UV (> or =1 MED) emitted by a SSR source we have noticed that: (a) neutrophils are initially the main infiltrating cell type in the dermis and (b) these infiltrating cells are the a key source of in vivo enzymatically [corrected] active enzymes such as elastase, [corrected] matrix metallo proteinases-1 and -9 (MMPs-1 and -9). These enzymes are relevant to the process of photoaging, as they break down the extracellular matrix. Keratinocytes and fibroblasts also produce matrix degrading enzymes, but to a lesser extent. Our results indicate a primary role for infiltrating neutrophils in the initial steps of photoaging. This is further supported by the observation that after exposure of skin type VI to physical doses of SSR, equivalent to those used for skin types I-III, no neutrophils and neutrophil-derived enzymatic activity were observed, explaining why skin type VI is [corrected] less susceptible to photoaging than skin types [corrected] I-III. Statement: Although most of the data, referred to, have been published, the current perspective in which they are put together is completely novel and has not been published elsewhere.

Improving radiation-damage substructures for RIP.

Specific radiation damage can be used to solve macromolecular structures using the radiation-damage-induced phasing (RIP) method. The method has been investigated for six disulfide-containing test structures (elastase, insulin, lysozyme, ribonuclease A, trypsin and thaumatin) using data sets that were collected on a third-generation synchrotron undulator beamline with a highly attenuated beam. Each crystal was exposed to the unattenuated X-ray beam between the collection of a 'before' and an 'after' data set. The X-ray 'burn'-induced intensity differences ranged from 5 to 15%, depending on the protein investigated. X-ray-susceptible substructures were determined using the integrated direct and Patterson methods in SHELXD. The best substructures were found by downscaling the 'after' data set in SHELXC by a scale factor K, with optimal values ranging from 0.96 to 0.99. The initial substructures were improved through iteration with SHELXE by the addition of negatively occupied sites as well as a large number of relatively weak sites. The final substructures ranged from 40 to more than 300 sites, with strongest peaks as high as 57sigma. All structures except one could be solved: it was not possible to find the initial substructure for ribonuclease A, however, SHELXE iteration starting with the known five most susceptible sites gave excellent maps. Downscaling proved to be necessary for the solution of elastase, lysozyme and thaumatin and reduced the number of SHELXE iterations in the other cases. The combination of downscaling and substructure iteration provides important benefits for the phasing of macromolecular structures using radiation damage.

On the influence of the incident photon energy on the radiation damage in crystalline biological samples.

Two series of complete and highly redundant data sets were collected at wavelengths of 1.00 and 2.00 Angstroms on a cadmium derivative of porcine pancreatic elastase (PPE). Radiation damage to the sample was evaluated qualitatively by inspecting consecutive difference electron density maps during the course of the experiment. The nature of the radiation damage was found to be identical at both wavelengths and was localized primarily at the four disulfide bridges of PPE, the cadmium site and the two methionine residues. For a quantitative examination of the radiation damage, the decrease in the peak height of the cadmium ion in various electron density maps was exploited. Again, no significant difference in radiation damage between the two wavelengths was observed. This can be rationalized by considering the wavelength dependencies of the number of diffracted photons versus the number of absorbed photons and the energy deposited in the crystal by the latter.

UVA light stimulates the production of cathepsin G and elastase-like enzymes by dermal fibroblasts: a possible contribution to the remodeling of elastotic areas in sun-damaged skin.

Solar elastosis is characterized by accumulation of large amounts of material staining similarly to elastin in the dermis. The nature of this material and the process responsible for its accumulation are still unknown. Elastolytic proteases have important functions in the catabolism of the interstitial matrix and can also generate, by the digestion of the interstitial proteins, soluble peptides which can induce collagen and elastin synthesis and deposition. We investigated whether (i) elastolytic enzymes can be detected in samples from sun-exposed and non-exposed skin, and (ii) ultraviolet (UV) rays influence the production of elastolytic activities in cultured dermal fibroblasts. Immunoelectron microscopy showed a positive reaction for neutrophil elastase and cathepsin G in fibroblast-like cells from specimens of sun-exposed areas. Little or no reaction was found in biopsies of sun-protected skin. Fibroblast cultures from sun-exposed skin expressed higher levels of hydrolytic activity against synthetic substrates of elastases and cathepsin G than those obtained from sun-protected areas. Irradiation with UVA strongly stimulated the production of these activities in fibroblasts from sun-protected sites. No significant change was detected in parallel sets of cultures after UVB irradiation. Inhibition experiments indicated that the elastase-like activity expressed by fibroblasts can be attributed to at least two enzymes.

[Physico-chemical and biological characteristics of Pseudomonas aeruginosa elastase]. / Nekotorye fiziko-khimicheskie i biologicheskie kharakteristiki élastazy Pseudomonas aeruginosa.

Elastase isolated from P. aeruginosa clinical strain hydrolyzes elastin, casein, hemoglobin, ovalbumin, gelatin, fibrin, collagen. The optimum pH ensuring the activity of the enzyme is 7.8-8.0. Elastase shows maximum stability at pH 6.6-9.0. Heating at 80 degrees C for 10 minutes results in its practically complete inactivation. Elastase is a highly radiosensitive enzyme. Chelating agents and zinc, cobalt, mercury ions suppress its activity. Sodium and ammonium chlorides selectively inhibit the elastolytic, but not proteolytic activity of the enzyme. Elastase shows pronounced dermonecrotic and keratolytic action.