Response of Burkholderia cenocepacia H111 to micro-oxia.

B. cenocepacia is an opportunistic human pathogen that is particularly problematic for patients suffering from cystic fibrosis (CF). In the CF lung bacteria grow to high densities within the viscous mucus that is limited in oxygen. Pseudomonas aeruginosa, the dominant pathogen in CF patients, is known to grow and survive under oxygen-limited to anaerobic conditions by using micro-oxic respiration, denitrification and fermentative pathways. In contrast, inspection of the genome sequences of available B. cenocepacia strains suggested that B. cenocepacia is an obligate aerobic and non-fermenting bacterium. In accordance with the bioinformatics analysis we observed that B. cenocepacia H111 is able to grow with as little as 0.1% O2 but not under strictly anoxic conditions. Phenotypic analyses revealed that H111 produced larger amounts of biofilm, pellicle and proteases under micro-oxic conditions (0.5%-5% O2, i.e. conditions that mimic those encountered in CF lung infection), and was more resistant to several antibiotics. RNA-Seq and shotgun proteomics analyses of cultures of B. cenocepacia H111 grown under micro-oxic and aerobic conditions showed up-regulation of genes involved in the synthesis of the exopolysaccharide (EPS) cepacian as well as several proteases, two isocitrate lyases and other genes potentially important for life in micro-oxia. DATA DEPOSITION: RNA-Seq raw data files are accessible through the GEO Series accession number GSE48585. MS data have been deposited in the ProteomeXchange database (PXD000270).

Bioinspired surfaces against bacterial infections.

Bioactive surfaces that can prevent bacterial infections are of great interest since device-related infections represent an emerging and major threat to our health-care system. For that purpose, dopamine and its derivatives have been shown to provide an exceptionally facile method for the immobilization of bioactive compounds on metal oxide surfaces through an operationally simple dip-and-rinse procedure. Based on this method, three different coating strategies for the assembly of antifouling, antibacterial, and quorum sensing modulating surfaces have been established. The anachelin chromophore and nitro-dopamine proved to be very suitable anchoring moieties as they possess better oxidative stability and binding efficiency compared to dopamine itself. Furthermore, adsorbed bioactive hybrids were shown to be stable and recycling of the surfaces could be achieved. These examples clearly demonstrate the efficiency of this approach for the production of active surfaces, such as in biomedical devices.

Fluorescent labeling agents for quorum-sensing receptors (FLAQS) in live cells.

Lighten up: A selective fluorescent-labeling agent for quorum sensing (FLAQS) can be used for the visualization of the communication pathway of bacteria in live cells (see figure). This represents a new, operationally simple, fast, and inexpensive tool for the imaging of quorum-sensing receptors by using fluorescently labeled signaling-molecule analogues.

Bioinspired, releasable quorum sensing modulators.

We demonstrate the synthesis and immobilization of natural product hybrids featuring an acyl-homoserine lactone and a nitrodopamine onto biocompatible TiO(2) surfaces through an operationally simple dip-and-rinse procedure. The resulting immobilized hybrids were shown to be powerful quorum sensing (QS) activators in Pseudomonas strains acting by slow release from the surface.

The AHL- and BDSF-dependent quorum sensing systems control specific and overlapping sets of genes in Burkholderia cenocepacia H111.

Quorum sensing in Burkholderia cenocepacia H111 involves two signalling systems that depend on different signal molecules, namely N-acyl homoserine lactones (AHLs) and the diffusible signal factor cis-2-dodecenoic acid (BDSF). Previous studies have shown that AHLs and BDSF control similar phenotypic traits, including biofilm formation, proteolytic activity and pathogenicity. In this study we mapped the BDSF stimulon by RNA-Seq and shotgun proteomics analysis. We demonstrate that a set of the identified BDSF-regulated genes or proteins are also controlled by AHLs, suggesting that the two regulons partially overlap. The detailed analysis of two mutually regulated operons, one encoding three lectins and the other one encoding the large surface protein BapA and its type I secretion machinery, revealed that both AHLs and BDSF are required for full expression, suggesting that the two signalling systems operate in parallel. In accordance with this, we show that both AHLs and BDSF are required for biofilm formation and protease production.

Different protein expression profiles in cheese and clinical isolates of Enterococcus faecalis revealed by proteomic analysis.

The use of Enterococcus faecalis in the food industry has come under dispute because of the pathogenic potential of some strains of this species. In this study, we have compared the secretome and whole-cell proteome of one food isolate (E. faecalis DISAV 1022) and one clinical isolate (E. faecalis H1) by 2-DE and iTRAQ analyses, respectively. Extracellular protein patterns differed significantly, with only seven proteins common to both strains. Notably, only the clinical isolate expressed various well-characterized virulence factors such as the gelatinase coccolysin (GelE) and the extracellular serine proteinase V8 (SprE). Moreover, various other putative virulence factors, e.g. superoxide dismutase, choline- and chitin-binding proteins and potential moonlighting proteins, have been detected exclusively in the secretome of the clinical isolate, but not in the food isolate. The iTRAQ analysis of whole-cell proteins of the two strains highlighted a stronger expression of pathogenic traits such as an endocarditis-specific antigen and an adhesion lipoprotein in the pathogenic strain E. faecalis H1. Subsequently, six food isolates (including E. faecalis DISAV 1022) and six clinical isolates (including E. faecalis H1) were tested for the presence of gelatinase and protease activity in the culture supernatants. Both enzymatic activities were found in the clinical as well as the food isolates which clearly indicates that protease expression is strain specific and not representative for pathogenic isolates. Genetic analyses revealed that not only the gelatinase and serine protease genes but also the regulatory fsr genes must be present to allow protease expression.

A proteomics approach to study synergistic and antagonistic interactions of the fungal-bacterial consortium Fusarium oxysporum wild-type MSA 35.

Fusarium oxysporum is an important plant pathogen that causes severe damage of many economically important crop species. Various microorganisms have been shown to inhibit this soil-borne plant pathogen, including non-pathogenic F. oxysporum strains. In this study, F. oxysporum wild-type (WT) MSA 35, a biocontrol multispecies consortium that consists of a fungus and numerous rhizobacteria mainly belonging to gamma-proteobacteria, was analyzed by two complementary metaproteomic approaches (2-DE combined with MALDI-Tof/Tof MS and 1-D PAGE combined with LC-ESI-MS/MS) to identify fungal or bacterial factors potentially involved in antagonistic or synergistic interactions between the consortium members. Moreover, the proteome profiles of F. oxysporum WT MSA 35 and its cured counter-part CU MSA 35 (WT treated with antibiotics) were compared with unravel the bacterial impact on consortium functioning. Our study presents the first proteome mapping of an antagonistic F. oxysporum strain and proposes candidate proteins that might play an important role for the biocontrol activity and the close interrelationship between the fungus and its bacterial partners.

A gel-free quantitative proteomics approach to investigate temperature adaptation of the food-borne pathogen Cronobacter turicensis 3032.

The opportunistic food-borne pathogen Cronobacter sp. causes rare but significant illness in neonates and is capable to grow at a remarkably wide range of temperatures from 5.5 to 47 degrees C. A gel-free quantitative proteomics approach was employed to investigate the molecular basis of the Cronobacter sp. adaptation to heat and cold-stress. To this end the model strain Cronobacter turicensis 3032 was grown at 25, 37, 44, and 47 degrees C, and whole-cell and secreted proteins were iTRAQ-labelled and identified/quantified by 2-D-LC-MALDI-TOF/TOF-MS. While 44 degrees C caused only minor changes in C. turicensis growth rate and protein profile, 47 degrees C affected the expression of about 20% of all 891 identified proteins and resulted in a reduced growth rate and rendered the strain non-motile and filamentous. Among the heat-induced proteins were heat shock factors, transcriptional and translational proteins, whereas proteins affecting cellular morphology, proteins involved in motility, central metabolism and energy production were down-regulated. Notably, numerous potential virulence factors were found to be up-regulated at higher temperatures, suggesting an elevated pathogenic potential of Cronobacter sp. under these growth conditions. Significant alterations in the protein expression profile and growth rate of C. turicensis exposed to 25 degrees C indicate that at this temperature the organism is cold-stressed. Up-regulated gene products comprised cold-shock, DNA-binding and ribosomal proteins, factors that support protein folding and proteins opposing cold-induced decrease in membrane fluidity, whereas down-regulated proteins were mainly involved in central metabolism.