Damien Seyer

Identification of Biofilm-Associated Cluster (bac) in Pseudomonas aeruginosa Involved in Biofilm Formation and Virulence

Biofilms are prevalent in diseases caused by Pseudomonas aeruginosa, an opportunistic and nosocomial pathogen. By a proteomic approach, we previously identified a hypothetical protein of P. aeruginosa (coded by the gene pA3731) that was accumulated by biofilm cells. We report here that a ΔpA3731 mutant is highly biofilm-defective as compared with the wild-type strain. Using a mouse model of lung infection, we show that the mutation also induces a defect in bacterial growth during the acute phase of infection and an attenuation of the virulence. The pA3731 gene is found to control positively the ability to swarm and to produce extracellular rhamnolipids, and belongs to a cluster of 4 genes (pA3729–pA3732) not previously described in P. aeruginosa. Though the protein PA3731 has a predicted secondary structure similar to that of the Phage Shock Protein, some obvious differences are observed compared to already described psp systems, e.g., this unknown cluster is monocistronic and no homology is found between the other proteins constituting this locus and psp proteins. As E. coli PspA, the amount of the protein PA3731 is enlarged by an osmotic shock, however, not affected by a heat shock. We consequently named this locus bac for biofilm-associated cluster.

Peroxiredoxin 2 is involved in the neuroprotective effects of PACAP in cultured cerebellar granule neurons.

The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) is known to counteract in vitro the deleterious effects of toxic agents on cerebellar granule cell survival and differentiation. The potent antiapoptotic action of PACAP is mediated through inhibition of caspase-3 activity; however, additional proteins are likely involved and remain to be identified. Two-dimensional gel electrophoresis analysis coupled with mass spectrometry characterization led to the identification of a protein, peroxiredoxin 2, which was induced after a 6-h treatment with PACAP. Western blot analysis confirmed the regulation of peroxiredoxin 2 by PACAP and revealed that this protein is induced by both cyclic AMP and protein kinase C stimulators. Inhibition of peroxiredoxin 2 expression, using two distinct small-interfering RNAs (siRNAs), reduced the effect of PACAP on caspase-3 activity and cerebellar granule cell survival. Peroxiredoxin 2 expression was also induced in vivo and in vitro by ethanol. Although ethanol and PACAP exert opposite effects on caspase-3 activity, inhibition of peroxiredoxin 2 expression, using siRNAs, only reduced the ability of PACAP to prevent ethanol-induced caspase-3 activity. Taken together, these data indicate that peroxiredoxin 2 is probably involved in the neurotrophic effect of PACAP and suggest that this protein may have a therapeutic potential for the treatment of some neurodegenerative diseases.

Proteomic comparison of outer membrane protein patterns of sessile and planktonic Pseudomonas aeruginosa cells

ABSTRACT We have compared the outer membrane protein (OMP) pattern of sessile Pseudomonas aeruginosa cells with that of its stationary-phase free-floatingcounterparts. Two-dimensional electrophoretic gels were compared usingcomputing scanning densitometry. The level of 103 OMPs (i.e. about 50% ofthe spots that fulfilled our statistical selection threshold) was significantly andreproducibly different in biofilm organisms as compared with their planktoniccounterparts. Some OMPs that specifically either accumulated or decreasedwere identified by mass spectrometry and the role of these proteins in thebiofilm phenotype is discussed. * Corresponding author:Dr P. CosetteUMR 6522 CNRSFaculty of SciencesUniversity of Rouen76821 Mont-Saint-Aignan CedexFrance T 33 235 146397 F 33 235 146704 E pascal.cosette@univ-rouen.fr 1 IBBR Group, Laboratory “Polymeres,`Biopolymeres, Membranes”, UMR`6522 CNRS, University of Rouen,France 2 Laboratory of Cellular and MolecularNeuroendocrinology, INSERMU-413, UA CNRS, University ofRouen, France

Bacterial Floras and Biofilms of Malignant Wounds Associated with Breast Cancers

ABSTRACT The risk of infections and the appearance of symptoms (e.g., odors) represent the main troubles resulting from malignant wounds. The aim of this study was to characterize the balance of bacterial floras and the relationships between biofilms and bacteria and the emergence of symptoms. Experimental research was carried out for 42 days on malignant wounds associated with breast cancer. Investigations of bacterial floras (aerobes, aero-anaerobes, and anaerobes), detection of the presence of biofilms by microscopic epifluorescence, and clinical assessment were performed. We characterized biofilms in 32 malignant wounds associated with breast cancer and bacterial floras in 25 such wounds. A mixed group of floras, composed of 54 different bacterial types, was identified, with an average number per patient of 3.6 aerobic species and 1.7 anaerobic species; the presence of strict anaerobic bacterial strains was evidenced in 70% of the wounds; biofilm was observed in 35% of the cases. Odor was a reliable indicator of colonization by anaerobes, even when this symptom was not directly linked to any of the identified anaerobic bacteria. Bacteria are more likely to be present during myelosuppression and significantly increase the emergence of odors and pain when present at amounts of >105 · g−1. The presence of biofilms was not associated with clinical signs or with precise types of bacteria. No infections occurred during the 42-day evaluation period. This study provides a dynamic description of the bacterial floras of tumoral wounds. The study results highlight the absolute need for new therapeutic options that are effective for use on circulating bacteria as well as on bacteria organized in biofilm.

An overview of techniques for the characterization and quantification of microbial colonization on stone monuments

Biodeterioration can be defined as any undesired change of the properties of a material caused by biological activity of living organisms. The biodeterioration of stone materials is related to the production of pigments (aesthetic action), to cell metabolism (biochemical action) and to the mechanical action of the biomass colonizing the material during its growth (physical action). Quantification of the sessile biomass and characterization of microbial communities colonizing stone are essential first steps to ensure the diagnosis of biodeterioration processes and to implement control strategies and appropriate treatment. Different destructive and non-destructive approaches can be used to sample stone specimens from monuments: scraping, swab using, and cutting. Different analytical methods can be used depending on the type of microorganism sought: determination of chlorophyll content and color analysis for pigmented microorganisms; measurement of in situ physiological activity of surface microcolonies by applying fluorogenic substrate analogues or confocal laser scanning microscopy observations after CTC staining for active biomass; scanning or transmission electron microscopy observation for biofilm visualization; enzyme-linked immunosorbent assay for the investigation of both microorganisms that can and cannot be cultured; classical microbiological methods, which consist in cultivation of microorganisms on synthetic media; and molecular methods for the study of microbial biodiversity based on the polymorphism of molecular markers using PCR, hybridization, classical or high throughput sequencing. The aim of this review is to present basics of the different biodeterioration mechanisms and to focus on the main techniques that can be used to characterize and quantify the biodeterioration biomass.

Identification of Proteins Regulated by PACAP in PC12 Cells by 2D Gel Electrophoresis Coupled to Mass Spectrometry

Abstract:  The rat pheochromocytoma PC12 cell line has been widely used as a model to study neuronal differentiation. In particular, after serum depletion, PC12 cells stop to proliferate and undergo apoptosis. Under such conditions, treatment with pituitary adenylate cyclase‐activating polypeptide (PACAP) promotes cell survival and induces neurite outgrowth. The identification of the proteins regulated by PACAP in PC12 cells under apoptotic conditions should provide valuable information concerning the mechanisms controlling neuronal cell survival and differentiation. To this aim, PC12 cells cultured in serum‐free medium were treated with PACAP (10−7 M), proteins were extracted, separated by two‐dimensional gel electrophoresis (2‐DE), and identified by MALDI‐ToF mass spectrometry. The comparison between 16 2‐DE maps led to the characterization of 110 proteins regulated by PACAP among which 22 have been identified by automatic query of the Mascot, Aldente, and Profound servers with the ProGeR‐CDD database. Seventy‐six percent of these proteins, including the p17 subunit of caspase‐3, the heat shock protein hsp60, and the GTPase ran were found to be repressed whereas the others notably hsp27, tubulin β‐5, and calmodulin were overexpressed. Investigation of the putative functions indicated that some of the proteins regulated by PACAP and identified in the present article could control cell survival or differentiation.

Kinetic development of biofilm on NF membranes at the Méry-sur-Oise plant, France

The kinetic formation of biofilms developing on nanofiltration (NF) membranes was studied for 2 years in the water production unit of Méry-sur-Oise, France. New membranes were set up in a pilot train integrated to the plant and autopsied after operation for 7, 80, 475 and 717 days. The biofouling layer was studied by confocal laser scanning microscope after 4′,6-diamidino-2-phenyindole dihydrochloride and lectin staining, and by attenuated total reflectance-Fourier transform infrared spectroscopy and rheology experiments. Three stages of biofilm growth were discriminated: (1) the presence of sessile microcolonies embedded in an exopolymeric matrix (after filtration for seven days); (2) membrane coverage expansion through microcolony development and biofilm growth in three dimensions (up to 80 days filtration); and (3) biofilm maturation by densification (after filtration for 80–717 days). Biofilm maturation resulted in total coverage of the membrane surface and matrix residue diversification, development of the polysaccharide network, and the strengthening of matrix cohesion through viscosity and elasticity increases. The wettability and permeability of the fouled NF membranes decreased quickly and continuously throughout the biofilm development process. The longitudinal pressure drop (LPD) increased only after the biofilm reached a quantitative threshold. The decline in membrane permeability may be the result of contributions from many fouling mechanisms but the LPD was more substantially influenced by biofilm development.

Synergistic antibiofilm efficacy of various commercial antiseptics, enzymes and EDTA: a study of Pseudomonas aeruginosa and Staphylococcus aureus biofilms

A multistep strategy was used to generate a combined antibiofilm treatment that could efficiently decrease the biomass of dense biofilms (≥6 × 10(7) CFU/cm(2)). Several compounds that exhibited activity against various targets were tested individually and in combination to search for possible synergistic effects. First, the antibiofilm activity of various commercially available antiseptics was tested on Pseudomonas aeruginosa and Staphylococcus aureus. Second, antiseptics were mixed with ethylene diamine tetra-acetic acid (EDTA), which is an ion chelator that can disturb biofilm organisation, and additive effects on biofilm biomass degradation were found for both strains. Then, enzymes with the ability to destabilise the biofilm matrix by hydrolysing either its proteins or its polysaccharides were used; as expected, they did not decrease bacterial viability but were revealed as efficient biomass reducers. The combination of antiseptics, EDTA and proteases, all at low concentrations, revealed a synergistic effect leading to total eradication of dense biofilms both of P. aeruginosa and S. aureus.

Elaboration of antibacterial plastic surfaces by a combination of antiadhesive and biocidal coatings of natural products

Antibacterial polyolefins surfaces, combining biocidal and antiadhesive properties, were elaborated by a covalent grafting of antimicrobial peptides (AMPs), able to kill adherent bacteria, on a pre-immobilized hyaluronic acid (HA) layer, able to repel the micro-organisms. Different HA activation rate for its immobilization, were used to change HA layer morphology and number of residual free carboxylic acid functions for AMPs grafting. Based on adhesion tests on Staphylococcus epidermidis and microscopy fluorescent observations, the presence of the two combined properties was shown to be depended on the HA activation rate. Thus, the best addition effect was observed for an AMP grafting on a surface based on a high HA activation, data pointing out a decrease of the bacterial adhesion up to 99.8% and a perturbation of the bacterial membrane integrity of adhered bacteria. On the contrary, a decrease of the antibacterial activity was observed for an AMP grafting on a surface based on a low HA activation.

Characterization of new outer membrane proteins of Pseudomonas aeruginosa using a combinatorial peptide ligand library.

Most often, the use of ProteoMiner beads has been restricted to human serum proteins for the normalization of major proteins, such as albumin. However, there are other situations of interest in which the presence of major proteins would quench the signals of low abundance polypeptides. We propose the use of these beads for investigating the envelope of the gram-negative bacterium Pseudomonas aeruginosa. Initially, we performed comparative 2D electrophoresis to qualitatively evaluate the incidence of the normalization stage. This demonstrated a significant reduction of the major membrane proteins. Thereafter, using shotgun analysis, the same protein extract was targeted by using combinatorial peptide ligand library capture. This treatment yielded 154 additional outer membrane proteins (OMPs) uncovered by the study of the crude sample.