Biomedical subjects

Leo Eberl

Publications and source records attributed to Leo Eberl.

At least 19 recordsLinked to original sources

PubMed2007

Microbial landscapes: new paths to biofilm research.

It is the best of times for biofilm research. Systems biology approaches are providing new insights into the genetic regulation of microbial functions, and sophisticated modelling techniques are enabling the prediction of microbial community structures. Yet it is also clear that there is a need for ecological theory to contribute to our understanding of biofilms. Here, we suggest a concept for biofilm research that is spatially explicit and solidly rooted in ecological theory, which might serve as a universal approach to the study of the numerous facets of biofilms.

Bacterial Physiological Phenomena↗

PubMed2006

Identification of bacterial N-acylhomoserine lactones (AHLs) with a combination of ultra-performance liquid chromatography (UPLC), ultra-high-resolution mass spectrometry, and in-situ biosensors.

N-Acylated homoserine lactones (AHLs) are produced by Gram-negative bacteria as communication signals and are frequently studied as mediators of the "quorum sensing" response of bacterial communities. Several reports have recently been published on the identification of AHLs from different species and attempts have been made to study their role in natural habitats, for example the surface of plant roots in the rhizosphere. In this article, different analytical methods, including bacterial biosensors and chromatographic techniques, are reviewed. A concept for assignment of the structures of AHLs is also presented. The retention behaviour of derivatives of AHLs containing beta-keto or hydroxyl groups and/or double bonds has been evaluated in relation to the separation behaviour of AHLs with saturated and unsubstituted alkanoyl chains. Samples have also been analysed by high resolution mass spectrometry (Fourier-transform ion-cyclotron-resonance mass spectrometry, FTICR-MS), nano liquid chromatography-electrospray ionization ion trap mass spectrometry (nano-LC-MS) and by the aid of a biosensor. The results obtained from ultra performance liquid chromatography (UPLC), FTICR-MS, nano-LC-MS, and bioassays have been compared to attempt structural characterisation of AHL without chemical synthesis of analytical standards. The method was used to identify the major AHL compound produced by the rhizosphere bacterium Acidovorax sp. N35 as N-(3-hydroxydecanoyl)homoserine lactone.

4-Butyrolactone↗

PubMed2006

The Burkholderia cepacia bceA gene encodes a protein with phosphomannose isomerase and GDP-D-mannose pyrophosphorylase activities.

The bceA gene is part of the Burkholderia cepacia IST408 exopolysaccharide (EPS) biosynthetic cluster. It encodes a 55.3-kDa bifunctional protein (type II PMI family) with phosphomannose isomerase (PMI) and GDP-mannose pyrophosphorylase (GMP) activities. GMP activity is strongly dependent on the presence of Ca(2+) or Mn(2+), while PMI activity can use a broader variety of divalent cations (Ca(2+)>Mn(2+)>Mg(2+)>Co(2+)>Ni(2+)). The lack of a functional bceA gene does not affect EPS production yield in a non-polar insertion bceA mutant. The in silico search for putative bceA homologues revealed the presence of 2-5 bceA orthologues in the Burkholderia genomes available. This suggests that in B. cepacia IST408 putative bceA functional homologues may compensate the bceA mutation. However, the viscosity of aqueous solutions prepared with the EPS produced by the bceA mutant was significantly reduced compared with wild-type biopolymer and the mutant forms biofilms with a size reduced by 6-fold.

Burkholderia cepacia↗

PubMed2006

Interactions between bacteria and eukaryotes via small molecules.

The interactions that occur between eukaryotes and bacteria have long been of interest, as knowledge of these processes could lead to the development of novel therapeutics and other potential applications in biotechnology. Many of these interactions are mediated by small molecules, which have subsequently formed the focus of numerous studies. An arsenal of small molecules exhibiting a wide range of activities has been isolated from various sources, including plants, animals and microorganisms. As a number of these compounds are pharmacologically active, there is a strong continued interest in natural product chemistry. Recent developments in this field have focused on two areas: evidence has been gathered to show that secondary metabolites are often produced by symbiotic bacteria, rather than by the eukaryotic host, and the importance of bacterial cell-to-cell signalling in bacteria-host interactions has been confirmed.

Bacterial Physiological Phenomena↗

PubMed2006

Molecular characterization of the alpha-glucosidase activity in Enterobacter sakazakii reveals the presence of a putative gene cluster for palatinose metabolism.

Enterobacter sakazakii is considered an opportunistic pathogen for premature infants and neonates. Although E. sakazakii has been isolated from various types of food, recontaminated dried infant formula has been epidemiologically identified as the major source of infection. Amongst others, alpha-glucosidase activity is one of the most important biochemical features, which differentiates E. sakazakii from other species in the family Enterobacteriaceae and has therefore been used as a selective marker in the development of differential media. However, it has been shown, that methods based on this biochemical feature are prone to producing false-positive results for presumptive E. sakazakii colonies due to the presence of this enzymatic activity in other species of the Enterobacteriaceae. Therefore, elucidation of the molecular basis responsible for the biochemical feature in E. sakazakii would provide novel targets suitable for the development of more specific and direct identification systems for this organism. By applying the bacterial artificial chromosome (BAC) approach, along with heterologous gene expression in Escherichia coli, the molecular basis of the alpha-glucosidase activity in E. sakazakii was characterized. Here we report the identification of two different alpha-glucosidase encoding genes. Homology searches of the deduced amino acid sequences revealed that the proteins belong to a cluster of gene products putatively responsible for the metabolism of isomaltulose (palatinose; 6-O-alpha-d-glucopyranosyl-d-fructose). The glycosyl-hydrolyzing activity of each protein was demonstrated by subcloning the respective open reading frames and screening of E. coli transformants for their ability to hydrolyze 4-methyl-umbelliferyl-alpha-d-glucoside. Analysis at the protein level revealed that both enzymes belong to the intracellular fraction of cell proteins. The presence of the postulated palatinose metabolism was proven by growth experiments using this sugar as a sole carbon source.

Bacterial Proteins↗

PubMed2006

Quorum sensing in the genus Burkholderia.

The genus Burkholderia contains over 30 species, many of which are important human pathogens. In addition to the primary pathogens Burkholderia pseudomallei and Burkholderia mallei, several species have emerged as opportunistic pathogens in persons suffering from cystic fibrosis (CF) and immunocompromised individuals. All Burkholderia species investigated so far employ quorum-sensing (QS) systems that rely on N-acyl-homoserine lactone (AHL) signal molecules to express certain phenotypic traits in a population density-dependent manner. Whilst many Burkholderia strains only contain the CepI/CepR QS system, which relies on C8-HSL, some strains, in particular isolates of B. pseudomallei and B. mallei, harbour multiple LuxI/LuxR homologues and produce numerous AHL signal molecules. Evidence has accumulated over the past few years that the QS systems operating in Burkholderia are crucial for full virulence in various animal models. However, only few QS-regulated functions required for virulence in the different infection models have so far been identified. Given the essential role of QS in the expression of pathogenic traits in Burkholderia these regulatory systems represent attractive targets for the development of novel therapeutics.

4-Butyrolactone↗

PubMed2006

Towards the proteome of Burkholderia cenocepacia H111: setting up a 2-DE reference map.

Polyphasic-taxonomic studies of the past decade have shown that the Burkholderia cepacia complex (Bcc) comprises at least nine species, which share a high degree of 16S rDNA (98-100%) sequence similarity but only moderate levels of DNA-DNA hybridization. Members of the Bcc are well known as opportunistic pathogens of plants, animals and humans but also as biocontrol and bioremediation agents. In this study intra-, surface-associated and extracellular proteins of B. cenocepacia H111, which was isolated from a cystic fibrosis patient, were examined by 2-DE coupled to MALDI-TOF MS. MS and MS/MS data were searched against a database comprising all currently available annotated proteins of genetically closely related strains. In total 642 proteins spots were successfully identified corresponding to 390 different protein species, which were classified into functional categories. The majority of these proteins could be linked to housekeeping functions in energy production, amino acid metabolism, protein folding, post-translational modification and turnover, and translation. Noteworthy is the fact that a significant number of truly secreted and membrane proteins were identified in the extracellular and surface-associated sub-proteomes. This indicates that the pre-fractionation protocol used in this study is a highly valuable strategy for unravelling the cellular location of the identified proteins.

Bacterial Proteins↗

PubMed2006

From a local dialect to a common language.

Quinolone-dependent cell-to-cell signaling was thought to be limited to Pseudomonas aeruginosa. The study by Diggle et al. in the July issue of Chemistry & Biology now presents clear evidence that quinolone signaling molecules are produced by several bacterial species and may be used for intergenus communication.

Pseudomonas aeruginosa↗

PubMed2006

Induction of systemic resistance in tomato by N-acyl-L-homoserine lactone-producing rhizosphere bacteria.

N-acyl-L-homoserine lactone (AHL) signal molecules are utilized by Gram-negative bacteria to monitor their population density (quorum sensing) and to regulate gene expression in a density-dependent manner. We show that Serratia liquefaciens MG1 and Pseudomonas putida IsoF colonize tomato roots, produce AHL in the rhizosphere and increase systemic resistance of tomato plants against the fungal leaf pathogen, Alternaria alternata. The AHL-negative mutant S. liquefaciens MG44 was less effective in reducing symptoms and A. alternata growth as compared to the wild type. Salicylic acid (SA) levels were increased in leaves when AHL-producing bacteria colonized the rhizosphere. No effects were observed when isogenic AHL-negative mutant derivatives were used in these experiments. Furthermore, macroarray and Northern blot analysis revealed that AHL molecules systemically induce SA- and ethylene-dependent defence genes (i.e. PR1a, 26 kDa acidic and 30 kDa basic chitinase). Together, these data support the view that AHL molecules play a role in the biocontrol activity of rhizobacteria through the induction of systemic resistance to pathogens.

4-Butyrolactone↗

PubMed2006

In situ quantitation of the spatial scale of calling distances and population density-independent N-acylhomoserine lactone-mediated communication by rhizobacteria colonized on plant roots.

We used computer-assisted microscopy at single cell resolution to quantify the in situ spatial scale of N-acylhomoserine lactone (AHL)-mediated cell-to-cell communication of Pseudomonas putida colonized on tomato and wheat root surfaces. The results of this in situ quantification study on close-to-natural surfaces challenge the conventional view of a quorum group requirement of high cell densities for this type of bacterial communication. In situ image analysis indicated that the effective 'calling distance' on root surfaces was most frequent at 4-5 microm, extended to 37 microm in the root tip/elongation zone and further out to 78 microm in the root hair zone. The spatial scale of these calling distances is very long-range in proportion to the size of individual bacteria. Geostatistical modeling analysis implicated the importance of AHL-gradients mediating effective communication between remote cells. We conclude that AHL-mediated cell-to-cell communication occurs not only within dense populations, but also in very small groups and over long ranges between individual bacteria, and therefore this cellular activity is more commonplace and effective than hitherto predicted. We propose that this cell-to-cell communication is governed more by the in situ spatial proximity of cells within AHL-gradients than the requirement for a quorum group of high population density.

4-Butyrolactone↗

PubMed2006

Computer-aided design of agents that inhibit the cep quorum-sensing system of Burkholderia cenocepacia.

Recent research has provided evidence that interference with bacterial cell-to-cell signaling is a promising strategy for the development of novel antimicrobial agents. Here we report on the computer-aided design of novel compounds that specifically inhibit an N-acyl-homoserine lactone-dependent communication system that is widespread among members of the genus Burkholderia. This genus comprises more than 30 species, many of which are important pathogens of animals and humans. Over the past few years, several Burkholderia species, most notably Burkholderia cenocepacia, have emerged as important opportunistic pathogens causing severe pulmonary deterioration in persons with cystic fibrosis. As efficient treatment of Burkholderia infections is hampered by the inherent resistance of the organisms to a large range of antibiotics, novel strategies for battling these pathogens need to be developed. Here we show that compounds targeting the B. cenocepacia signaling system efficiently inhibit the expression of virulence factors and attenuate the pathogenicity of the organism.

Anti-Bacterial Agents↗

PubMed2006

A quorum-quenching approach to investigate the conservation of quorum-sensing-regulated functions within the Burkholderia cepacia complex.

Taxonomic studies of the past few years have shown that the Burkholderia cepacia complex, a heterogeneous group of B. cepacia-like organisms, consists of at least nine species. B. cepacia complex strains are ubiquitously distributed in nature and have been used for biocontrol, bioremediation, and plant growth promotion purposes. At the same time, B. cepacia complex strains have emerged as important opportunistic pathogens of humans, particularly those with cystic fibrosis. All B. cepacia complex species investigated thus far use quorum-sensing (QS) systems that rely on N-acylhomoserine lactone (AHL) signal molecules to express certain functions, including the production of extracellular proteases, swarming motility, biofilm formation, and pathogenicity, in a population-density-dependent manner. In this study we constructed a broad-host-range plasmid that allowed the heterologous expression of the Bacillus sp. strain 240B1 AiiA lactonase, which hydrolyzes the lactone ring of various AHL signal molecules, in all described B. cepacia complex species. We show that expression of AiiA abolished or greatly reduced the accumulation of AHL molecules in the culture supernatants of all tested B. cepacia complex strains. Phenotypic characterization of wild-type and transgenic strains revealed that protease production, swarming motility, biofilm formation, and Caenorhabditis elegans killing efficiency was regulated by AHL in the large majority of strains investigated.

4-Butyrolactone↗

PubMed2006

Identification of Burkholderia pseudomallei genes required for the intracellular life cycle and in vivo virulence.

The bacterial pathogen Burkholderia pseudomallei invades host cells, escapes from endocytic vesicles, multiplies intracellularly, and induces the formation of actin tails and membrane protrusions, leading to direct cell-to-cell spreading. This study was aimed at the identification of B. pseudomallei genes responsible for the different steps of this intracellular life cycle. B. pseudomallei transposon mutants were screened for a reduced ability to form plaques on PtK2 cell monolayers as a result of reduced intercellular spreading. Nine plaque assay mutants with insertions in different open reading frames were selected for further studies. One mutant defective in a hypothetical protein encoded within the Bsa type III secretion system gene cluster was found to be unable to escape from endocytic vesicles after invasion but still multiplied within the vacuoles. Another mutant with a defect in a putative exported protein reached the cytoplasm but exhibited impaired actin tail formation in addition to a severe intracellular growth defect. In four mutants, the transposon had inserted into genes involved in either purine, histidine, or p-aminobenzoate biosynthesis, suggesting that these pathways are essential for intracellular growth. Three mutants with reduced plaque formation were shown to have gene defects in a putative cytidyltransferase, a putative lipoate-protein ligase B, and a hypothetical protein. All nine mutants proved to be significantly attenuated in a murine model of infection, with some mutants being essentially avirulent. In conclusion, we have identified a number of novel major B. pseudomallei virulence genes which are essential for the intracellular life cycle of this pathogen.

4-Aminobenzoic Acid↗

PubMed2006

Two GacA-dependent small RNAs modulate the quorum-sensing response in Pseudomonas aeruginosa.

In Pseudomonas aeruginosa, the GacS/GacA two-component system positively controls the quorum-sensing machinery and the expression of extracellular products via two small regulatory RNAs, RsmY and RsmZ. An rsmY rsmZ double mutant and a gacA mutant were similarly impaired in the synthesis of the quorum-sensing signal N-butanoyl-homoserine lactone, the disulfide bond-forming enzyme DsbA, and the exoproducts hydrogen cyanide, pyocyanin, elastase, chitinase (ChiC), and chitin-binding protein (CbpD). Both mutants showed increased swarming ability, azurin release, and early biofilm development.

Bacterial Proteins↗

PubMed2005

Diverse pathogenicity of Burkholderia cepacia complex strains in the Caenorhabditis elegans host model.

A fast screening method was developed to assess the pathogenicity of a diverse collection of environmental and clinical Burkholderia cepacia complex isolates in the nematode Caenorhabditis elegans. The method was validated by comparison with the standard slow-killing assay. We observed that the pathogenicity of B. cepacia complex isolates in C. elegans was strain-dependent but species-independent. The wide range of observed pathogenic phenotypes agrees with the high degree of phenotypic variation among species of the B. cepacia complex and suggests that the taxonomic classification of a given strain within the complex cannot predict pathogenicity.

Animals↗

PubMed2005

Discovery of complex mixtures of novel long-chain quorum sensing signals in free-living and host-associated marine alphaproteobacteria.

More than 100 bacterial isolates from various marine habitats were screened for AHL production by using gfp reporter constructs based on the lasR system of Pseudomonas aeruginosa and the luxR system of Vibrio fischeri. Of the 67 Alphaproteobacteria tested, most of which belonged into the so-called Roseobacter clade, 39 induced fluorescence in either one or both sensor strains up to 103-fold compared to controls. Acylated homoserine lactones were identified by GC-MS analysis and shown to have chain lengths of C8, C10, C13-C16, and C18. One or two double bonds were often present, while a keto or hydroxyl group occurred only rarely in the side chain. Most strains produced several different AHLs. C18-en-HSL and C18-dien-HSL were produced by Dinoroseobacter shibae, an aerobic anoxygenic phototrophic bacterium isolated from dinoflagellates, and are among the longest AHLs found to date. Z7-C14-en-HSL, which has previously been detected in Rhodobacter sphaeroides, was produced by Roseovarius tolerans and Jannaschia helgolandensis. This signal molecule was synthesised and shown to induce a similar response to the culture supernatant in the respective sensor strain. The widespread occurrence of quorum-sensing compounds in marine Alphaproteobacteria, both free-living strains and those associated to eukaryotic algae, points to a great importance of this signalling mechanism for the adaptation of the organisms to their widely different ecological niches.

Alphaproteobacteria↗

PubMed2005

Biofilm formation of Pseudomonas putida IsoF: the role of quorum sensing as assessed by proteomics.

Pseudomonas putida strains are frequently isolated from the rhizosphere of plants and many strains promote plant-growth, exhibit antagonistic activities against plant pathogens and have the capacity to degrade pollutants. Factors that appear to contribute to the rhizosphere fitness are the ability of the organism to form biofilms and the utilization of cell-to-cell-communication systems (quorum sensing, QS) to co-ordinate the expression of certain phenotypes in a cell density dependent manner. Recently, the ppu QS locus of the tomato rhizosphere isolate P. putida Iso F was characterized and an isogenic QS-negative ppuI mutant P. putida F117 was generated. In the present study we investigated the impact of QS and biofilm formation on the protein profile of surface-associated proteins of P. putida IsoF. This was accomplished by comparative proteome analyses of the P. putida wild type IsoF and the QS-deficient mutant F117 grown either in planktonic cultures or in 60 h old mature biofilms. Differentially expressed proteins were identified by peptide mass fingerprinting and database search in the completed P. putida KT2440 genome sequence. The sessile life style affected 129 out of 496 surface proteins, suggesting that a significant fraction of the bacterial genome is involved in biofilm physiology. In surface-attached cells 53 out of 484 protein spots were controlled by the QS system, emphasizing its importance as global regulator of gene expression in P. putida IsoF. Most interestingly, the impact of QS was dependent on whether cells were grown on a surface or in suspension; about 50% of the QS-controlled proteins identified in planktonic cultures were found to be oppositely regulated when the cells were grown as biofilms. Fifty-seven percent of all identified surface-controlled proteins were also regulated by the ppu QS system. In conclusion, our data provide strong evidence that the set of QS-regulated proteins overlaps substantially with the set of proteins differentially expressed in sessile cells.

Bacterial Proteins↗

PubMed2005

Identity and effects of quorum-sensing inhibitors produced by Penicillium species.

Quorum sensing (QS) communication systems are thought to afford bacteria with a mechanism to strategically cause disease. One example is Pseudomonas aeruginosa, which infects immunocompromised individuals such as cystic fibrosis patients. The authors have previously documented that blockage of the QS systems not only attenuates Ps. aeruginosa but also renders biofilms highly susceptible to treatment with conventional antibiotics. Filamentous fungi produce a battery of secondary metabolites, some of which are already in clinical use as antimicrobial drugs. Fungi coexist with bacteria but lack active immune systems, so instead rely on chemical defence mechanisms. It was speculated that some of these secondary metabolites could interfere with bacterial QS communication. During a screening of 100 extracts from 50 Penicillium species, 33 were found to produce QS inhibitory (QSI) compounds. In two cases, patulin and penicillic acid were identified as being biologically active QSI compounds. Their effect on QS-controlled gene expression in Ps. aeruginosa was verified by DNA microarray transcriptomics. Similar to previously investigated QSI compounds, patulin was found to enhance biofilm susceptibility to tobramycin treatment. Ps. aeruginosa has developed QS-dependent mechanisms that block development of the oxidative burst in PMN neutrophils. Accordingly, when the bacteria were treated with either patulin or penicillic acid, the neutrophils became activated. In a mouse pulmonary infection model, Ps. aeruginosa was more rapidly cleared from the mice that were treated with patulin compared with the placebo group.

Animals↗