Burkholderia cenocepacia H111 Produces a Water-Insoluble Exopolysaccharide in Biofilm: Structural Determination and Molecular Modelling.

Biofilms are a multicellular way of life, where bacterial cells are close together and embedded in a hydrated macromolecular matrix which offers a number of advantages to the cells. Extracellular polysaccharides play an important role in matrix setup and maintenance. A water-insoluble polysaccharide was isolated and purified from the biofilm produced by Burkholderia cenocepacia strain H111, a cystic fibrosis pathogen. Its composition and glycosidic linkages were determined using Gas-Liquid Chromatography-Mass Spectrometry (GLC-MS) on appropriate carbohydrate derivatives while its complete structure was unraveled by 1D and 2D NMR spectroscopy in deuterated sodium hydroxide (NaOD) aqueous solutions. All the collected data demonstrated the following repeating unit for the water-insoluble B. cenocepacia biofilm polysaccharide: [3)-α-d-Galp-(1→3)-α-d-Glcp-(1→3)-α-d-Galp-(1→3)-α-d-Manp-(1→]n Molecular modelling was used, coupled with NMR Nuclear Overhauser Effect (NOE) data, to obtain information about local structural motifs which could give hints about the polysaccharide insolubility. Both modelling and NMR data pointed at restricted dynamics of local conformations which were ascribed to the presence of inter-residue hydrogen bonds and to steric restrictions. In addition, the good correlation between NOE data and calculated interatomic distances by molecular dynamics simulations validated potential energy functions used for calculations.

High intracellular c-di-GMP levels antagonize quorum sensing and virulence gene expression in Burkholderia cenocepacia H111.

The opportunistic human pathogen Burkholderia cenocepacia H111 uses two chemically distinct signal molecules for controlling gene expression in a cell density-dependent manner: N-acyl-homoserine lactones (AHLs) and cis-2-dodecenoic acid (BDSF). Binding of BDSF to its cognate receptor RpfR lowers the intracellular c-di-GMP level, which in turn leads to differential expression of target genes. In this study we analysed the transcriptional profile of B. cenocepacia H111 upon artificially altering the cellular c-di-GMP level. One hundred and eleven genes were shown to be differentially expressed, 96 of which were downregulated at a high c-di-GMP concentration. Our analysis revealed that the BDSF, AHL and c-di-GMP regulons overlap for the regulation of 24 genes and that a high c-di-GMP level suppresses expression of AHL-regulated genes. Phenotypic analyses confirmed changes in the expression of virulence factors, the production of AHL signal molecules and the biosynthesis of different biofilm matrix components upon altered c-di-GMP levels. We also demonstrate that the intracellular c-di-GMP level determines the virulence of B. cenocepacia to Caenorhabditis elegans and Galleria mellonella.

In-Frame and Unmarked Gene Deletions in Burkholderia cenocepacia via an Allelic Exchange System Compatible with Gateway Technology.

Burkholderia cenocepacia is an emerging opportunistic pathogen causing life-threatening infections in immunocompromised individuals and in patients with cystic fibrosis, which are often difficult, if not impossible, to treat. Understanding the genetic basis of virulence in this emerging pathogen is important for the development of novel treatment regimes. Generation of deletion mutations in genes predicted to encode virulence determinants is fundamental to investigating the mechanisms of pathogenesis. However, there is a lack of appropriate selectable and counterselectable markers for use in B. cenocepacia, making its genetic manipulation problematic. Here we describe a Gateway-compatible allelic exchange system based on the counterselectable pheS gene and the I-SceI homing endonuclease. This system provides efficiency in cloning homology regions of target genes and allows the generation of precise and unmarked gene deletions in B. cenocepacia. As a proof of concept, we demonstrate its utility by deleting the Bcam1349 gene, encoding a cyclic di-GMP (c-di-GMP)-responsive regulator protein important for biofilm formation.

Regulation of biofilm formation in Pseudomonas and Burkholderia species.

In the present review, we describe and compare the molecular mechanisms that are involved in the regulation of biofilm formation by Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas aeruginosa and Burkholderia cenocepacia. Our current knowledge suggests that biofilm formation is regulated by cyclic diguanosine-5'-monophosphate (c-di-GMP), small RNAs (sRNA) and quorum sensing (QS) in all these bacterial species. The systems that employ c-di-GMP as a second messenger regulate the production of exopolysaccharides and surface proteins which function as extracellular matrix components in the biofilms formed by the bacteria. The systems that make use of sRNAs appear to regulate the production of exopolysaccharide biofilm matrix material in all these species. In the pseudomonads, QS regulates the production of extracellular DNA, lectins and biosurfactants which all play a role in biofilm formation. In B.cenocepacia QS regulates the expression of a large surface protein, lectins and extracellular DNA that all function as biofilm matrix components. Although the three regulatory systems all regulate the production of factors used for biofilm formation, the molecular mechanisms involved in transducing the signals into expression of the biofilm matrix components differ between the species. Under the conditions tested, exopolysaccharides appears to be the most important biofilm matrix components for P.aeruginosa, whereas large surface proteins appear to be the most important biofilm matrix components for P.putida, P.fluorescens, and B.cenocepacia.

The CRP/FNR family protein Bcam1349 is a c-di-GMP effector that regulates biofilm formation in the respiratory pathogen Burkholderia cenocepacia.

Burkholderia cenocepacia is an opportunistic respiratory pathogen that can cause severe infections in immune-compromised individuals and is associated with poor prognosis for patients suffering from cystic fibrosis. The second messenger cyclic diguanosine monophosphate (c-di-GMP) has been shown to control a wide range of functions in bacteria, but little is known about these regulatory mechanisms in B. cenocepacia. Here we investigated the role that c-di-GMP plays in the regulation of biofilm formation and virulence in B. cenocepacia. Elevated intracellular levels of c-di-GMP promoted wrinkly colony, pellicle and biofilm formation in B. cenocepacia. A screen for transposon mutants unable to respond to elevated levels of c-di-GMP led to the identification of the mutant bcam1349 that did not display increased biofilm and pellicle formation with excessive c-di-GMP levels, and displayed a biofilm defect with physiological c-di-GMP levels. The bcam1349 gene is predicted to encode a transcriptional regulator of the CRP/FNR superfamily. Analyses of purified Bcam1349 protein and truncations demonstrated that it binds c-di-GMP in vitro. The Bcam1349 protein was shown to regulate the production of a number of components, including cellulose and fimbriae. It was demonstrated that the Bcam1349 protein binds to the promoter region of the cellulose synthase genes, and that this binding is enhanced by the presence of c-di-GMP. The bcam1349 mutant showed reduced virulence in a Galleria mellonella wax moth larvae infection model. Taken together, these findings suggest that the Bcam1349 protein is a transcriptional regulator that binds c-di-GMP and regulates biofilm formation and virulence in B. cenocepacia in response to the level of c-di-GMP.

The BDSF quorum sensing receptor RpfR regulates Bep exopolysaccharide synthesis in Burkholderia cenocepacia via interaction with the transcriptional regulator BerB.

The polysaccharide Bep is essential for in vitro biofilm formation of the opportunistic pathogen Burkholderia cenocepacia. We found that the Burkholderia diffusible signaling factor (BDSF) quorum sensing receptor RpfR is a negative regulator of the bep gene cluster in B. cenocepacia. An rpfR mutant formed wrinkled colonies, whereas additional mutations in the bep genes or known bep regulators like berA and berB restored the wild-type smooth colony morphology. We found that there is a good correlation between intracellular c-di-GMP levels and bep expression when the c-di-GMP level is increased or decreased through ectopic expression of a diguanylate cyclase or a c-di-GMP phosphodiesterase, respectively. However, when the intracellular c-di-GMP level is changed by site directed mutagenesis of the EAL or GGDEF domain of RpfR there is no correlation between intracellular c-di-GMP levels and bep expression. Except for rpfR, deletion mutants of all 25 c-di-GMP phosphodiesterase and diguanylate cyclase genes encoded by B. cenocepacia showed no change to berA and bep gene expression. Moreover, bacterial two-hybrid assays provided evidence that RpfR and BerB physically interact and give specificity to the regulation of the bep genes. We suggest a model where RpfR binds BerB at low c-di-GMP levels to sequester this RpoN-dependent activator to an RpfR/RpfF complex. If the c-di-GMP levels rise, possibly by the enzymatic action of RpfR, BerB binds c-di-GMP and is released from the RpfR/RpfF complex and associates with RpoN to activate transcription of berA, and the BerA protein subsequently activates transcription of the bep genes.

Influence of putative exopolysaccharide genes on Pseudomonas putida KT2440 biofilm stability.

We report a study of the role of putative exopolysaccharide gene clusters in the formation and stability of Pseudomonas putida KT2440 biofilm. Two novel putative exopolysaccharide gene clusters, pea and peb, were identified, and evidence is provided that they encode products that stabilize P. putida KT2440 biofilm. The gene clusters alg and bcs, which code for proteins mediating alginate and cellulose biosynthesis, were found to play minor roles in P. putida KT2440 biofilm formation and stability under the conditions tested. A P. putida KT2440 derivative devoid of any identifiable exopolysaccharide genes was found to form biofilm with a structure similar to wild-type biofilm, but with a stability lower than that of wild-type biofilm. Based on our data, we suggest that the formation of structured P. putida KT2440 biofilm can occur in the absence of exopolysaccharides; however, exopolysaccharides play a role as structural stabilizers.

Quantitative analysis of the cellular inflammatory response against biofilm bacteria in chronic wounds.

Chronic wounds are an important problem worldwide. These wounds are characterized by a persistent inflammatory stage associated with excessive accumulation and elevated cell activity of neutrophils, suggesting that there must be a persistent stimulus that attracts and recruits neutrophils to the wound. One such stimulus might be the presence of bacterial biofilms in chronic wounds. In the present study, biopsy specimens from chronic venous leg ulcers were investigated for the detection of bacteria using peptide nucleic acid-based fluorescence in situ hybridization (PNA-FISH) and confocal laser scanning microscopy. The bacteria in the wounds were often situated in large aggregates. To obtain a measure of the cellular inflammatory response against the bacteria in the chronic wounds, the amount of neutrophils accumulated at the site of infection was evaluated through differential neutrophil counting on the tissue sections from wounds containing either Pseudomonas aeruginosa or Staphylococcus aureus. The P. aeruginosa-containing wounds had significantly higher numbers of neutrophils accumulated compared with the S. aureus-containing wounds. These results are discussed in relation to the hypothesis that the presence of P. aeruginosa biofilms in chronic wounds may be one of the main factors leading to a persistent inflammatory response and impaired wound healing.

The biofilm of Burkholderia cenocepacia H111 contains an exopolysaccharide composed of l-rhamnose and l-mannose: Structural characterization and molecular modelling.

Burkholderia cenocepacia belongs to the Burkholderia Cepacia Complex, a group of 22 closely related species both of clinical and environmental origin, infecting cystic fibrosis patients. B. cenocepacia accounts for the majority of the clinical isolates, comprising the most virulent and transmissible strains. The capacity to form biofilms is among the many virulence determinants of B. cenocepacia, a characteristic that confers enhanced tolerance to some antibiotics, desiccation, oxidizing agents, and host defenses. Exopolysaccharides are a major component of biofilm matrices, particularly providing mechanical stability to biofilms. Recently, a water-insoluble exopolysaccharide produced by B. cenocepacia H111 in biofilm was characterized. In the present study, a water-soluble exopolysaccharide was extracted from B. cenocepacia H111 biofilm, and its structure was determined by GLC-MS, NMR and ESI-MS. The repeating unit is a linear rhamno-tetrasaccharide with 50% replacement of a 3-α-L-Rha with a α-3-L-Man. [2)-α-L-Rhap-(1→3)-α-L-[Rhap or Manp]-(1→3)-α-L-Rhap-(1→2)-α-L-Rhap-(1→]n Molecular modelling was used to obtain information about local structural motifs which could give information about the polysaccharide conformation.

Nonrandom distribution of Pseudomonas aeruginosa and Staphylococcus aureus in chronic wounds.

The spatial organization of Pseudomonas aeruginosa and Staphylococcus aureus in chronic wounds was investigated in the present study. Wound biopsy specimens were obtained from patients diagnosed as having chronic venous leg ulcers, and bacterial aggregates in these wounds were detected and located by the use of peptide nucleic acid-based fluorescence in situ hybridization and confocal laser scanning microscopy (CLSM). We acquired CLSM images of multiple regions in multiple sections cut from five wounds containing P. aeruginosa and five wounds containing S. aureus and measured the distance of the bacterial aggregates to the wound surface. The distance of the P. aeruginosa aggregates to the wound surface was significantly greater than that of the S. aureus aggregates, suggesting that the distribution of the bacteria in the chronic wounds was nonrandom. The results are discussed in relation to our recent finding that swab culturing techniques may underestimate the presence of P. aeruginosa in chronic wounds and in relation to the hypothesis that P. aeruginosa bacteria located in the deeper regions of chronic wounds may play an important role in keeping the wounds arrested in a stage dominated by inflammatory processes.

Distribution, organization, and ecology of bacteria in chronic wounds.

Between 1 and 2% of the population in the developed world experiences a nonhealing or chronic wound characterized by an apparent arrest in a stage dominated by inflammatory processes. Lately, research groups have proposed that bacteria might be involved in and contribute to the lack of healing of these wounds. To investigate this, we collected and examined samples from chronic wounds obtained from 22 different patients, all selected because of suspicion of Pseudomonas aeruginosa colonization. These wound samples were investigated by standard culturing methods and peptide nucleic acid-based fluorescence in situ hybridization (PNA FISH) for direct identification of bacteria. By means of the culturing methods, Staphylococcus aureus was detected in the majority of the wounds, whereas P. aeruginosa was observed less frequently. In contrast, using PNA FISH, we found that a large fraction of the wounds contained P. aeruginosa. Furthermore, PNA FISH revealed the structural organization of bacteria in the samples. It appeared that P. aeruginosa aggregated as microcolonies imbedded in the matrix component alginate, which is a characteristic hallmark of the biofilm mode of growth. The present investigation suggests that bacteria present within these wounds tend to be aggregated in microcolonies imbedded in a self-produced matrix, characteristic of the biofilm mode of growth. Additionally, we must conclude that there exists no good correlation between bacteria detected by standard culturing methods and those detected by direct detection methods such as PNA FISH. This strongly supports the development of new diagnostic and treatment strategies for chronic wounds.

Key Players and Individualists of Cyclic-di-GMP Signaling in Burkholderia cenocepacia.

Burkholderia cenocepacia H111 is an opportunistic pathogen associated with chronic lung infections in cystic fibrosis patients. Biofilm formation, motility and virulence of B. cenocepacia are regulated by the second messenger cyclic di-guanosine monophosphate (c-di-GMP). In the present study, we analyzed the role of all 25 putative c-di-GMP metabolizing proteins of B. cenocepacia H111 with respect to motility, colony morphology, pellicle formation, biofilm formation, and virulence. We found that RpfR is a key regulator of c-di-GMP signaling in B. cenocepacia, affecting a broad spectrum of phenotypes under various environmental conditions. In addition, we identified Bcal2449 as a regulator of B. cenocepacia virulence in Galleria mellonella larvae. While Bcal2449 consists of protein domains that may catalyze both c-di-GMP synthesis and degradation, only the latter was essential for larvae killing, suggesting that a decreased c-di-GMP level mediated by the Bcal2449 protein is required for virulence of B. cenocepacia. Finally, our work suggests that some individual proteins play a role in regulating exclusively motility (CdpA), biofilm formation (Bcam1160) or both (Bcam2836).

Regulation of Burkholderia cenocepacia biofilm formation by RpoN and the c-di-GMP effector BerB.

Knowledge about the molecular mechanisms that are involved in the regulation of biofilm formation is essential for the development of biofilm-control measures. It is well established that the nucleotide second messenger cyclic diguanosine monophosphate (c-di-GMP) is a positive regulator of biofilm formation in many bacteria, but more knowledge about c-di-GMP effectors is needed. We provide evidence that c-di-GMP, the alternative sigma factor RpoN (σ54), and the enhancer-binding protein BerB play a role in biofilm formation of Burkholderia cenocepacia by regulating the production of a biofilm-stabilizing exopolysaccharide. Our findings suggest that BerB binds c-di-GMP, and activates RpoN-dependent transcription of the berA gene coding for a c-di-GMP-responsive transcriptional regulator. An increased level of the BerA protein in turn induces the production of biofilm-stabilizing exopolysaccharide in response to high c-di-GMP levels. Our findings imply that the production of biofilm exopolysaccharide in B. cenocepacia is regulated through a cascade involving two consecutive transcription events that are both activated by c-di-GMP. This type of regulation may allow tight control of the expenditure of cellular resources.

PNA-based fluorescence in situ hybridization for identification of bacteria in clinical samples.

Fluorescence in situ hybridization with PNA probes (PNA-FISH) that target specific bacterial ribosomal RNA sequences is a powerful and rapid tool for identification of bacteria in clinical samples. PNA can diffuse readily through the bacterial cell wall due to its uncharged backbone, and PNA-FISH can be performed with high specificity due to the extraordinary thermal stability of RNA-PNA hybrid complexes. We describe a PNA-FISH procedure and provide examples of the application of PNA-FISH for the identification of bacteria in chronic wounds, cystic fibrosis lungs, and soft tissue fillers. In all these cases, bacteria can be identified in biofilm aggregates, which may explain their recalcitrance to antibiotic treatment.

Colony morphology and transcriptome profiling of Pseudomonas putida KT2440 and its mutants deficient in alginate or all EPS synthesis under controlled matric potentials.

Pseudomonas putida is a versatile bacterial species adapted to soil and its fluctuations. Like many other species living in soil, P. putida often faces water limitation. Alginate, an exopolysaccharide (EPS) produced by P. putida, is known to create hydrated environments and alleviate the effect of water limitation. In addition to alginate, P. putida is capable of producing cellulose (bcs), putida exopolysaccharide a (pea), and putida exopolysaccharide b (peb). However, unlike alginate, not much is known about their roles under water limitation. Hence, in this study we examined the role of different EPS components under mild water limitation. To create environmentally realistic water limited conditions as observed in soil, we used the Pressurized Porous Surface Model. Our main hypothesis was that under water limitation and in the absence of alginate other exopolysaccharides would be more active to maintain homeostasis. To test our hypothesis, we investigated colony morphologies and whole genome transcriptomes of P. putida KT2440 wild type and its mutants deficient in synthesis of either alginate or all known EPS. Overall our results support that alginate is an important exopolysaccharide under water limitation and in the absence of alginate other tolerance mechanisms are activated.

The exopolysaccharide gene cluster Bcam1330-Bcam1341 is involved in Burkholderia cenocepacia biofilm formation, and its expression is regulated by c-di-GMP and Bcam1349.

In Burkholderia cenocepacia, the second messenger cyclic diguanosine monophosphate (c-di-GMP) has previously been shown to positively regulate biofilm formation and the expression of cellulose and type-I fimbriae genes through binding to the transcriptional regulator Bcam1349. Here, we provide evidence that cellulose and type-I fimbriae are not involved in B. cenocepacia biofilm formation in flow chambers, and we identify a novel Bcam1349/c-di-GMP-regulated exopolysaccharide gene cluster which is essential for B. cenocepacia biofilm formation. Overproduction of Bcam1349 in trans promotes wrinkly colony morphology, pellicle, and biofilm formation in B. cenocepacia. A screen for transposon mutants unable to respond to the overproduction of Bcam1349 led to the identification of a 12-gene cluster, Bcam1330-Bcam1341, the products of which appear to be involved in the production of a putative biofilm matrix exopolysaccharide and to be essential for flow-chamber biofilm formation. We demonstrate that Bcam1349 binds to the promoter region of genes in the Bcam1330-Bcam1341 cluster and that this binding is enhanced by the presence of c-di-GMP. Furthermore, we demonstrate that overproduction of both c-di-GMP and Bcam1349 leads to increased transcription of these genes, indicating that c-di-GMP and Bcam1349 functions together in regulating exopolysaccharide production from the Bcam1330-Bcam1341 gene cluster. Our results suggest that the product encoded by the Bcam1330-Bcam1341 gene cluster is a major exopolysaccharide that provides structural stability to the biofilms formed by B. cenocepacia, and that its production is regulated by c-di-GMP through binding to and promotion of the activity of the transcriptional regulator Bcam1349.

Biofilms in chronic infections - a matter of opportunity - monospecies biofilms in multispecies infections.

It has become evident that aggregation or biofilm formation is an important survival mechanism for bacteria in almost any environment. In this review, we summarize recent visualizations of bacterial aggregates in several chronic infections (chronic otitis media, cystic fibrosis, infection due to permanent tissue fillers and chronic wounds) both as to distribution (such as where in the wound bed) and organization (monospecies or multispecies microcolonies). We correlate these biofilm observations to observations of commensal biofilms (dental and intestine) and biofilms in natural ecosystems (soil). The observations of the chronic biofilm infections point toward a trend of low bacterial diversity and sovereign monospecies biofilm aggregates even though the infection in which they reside are multispecies. In contrast to this, commensal and natural biofilm aggregates contain multiple species that are believed to coexist, interact and form biofilms with high bacterial and niche diversity. We discuss these differences from both the diagnostic and the scientific point of view.

Successful resonance Raman study of cresolase activity of mushroom tyrosinase.

Mono-oxygenase (cresolase) activity of mushroom tyrosinase (MT) in the presence of 4-[(4-hydroxyphenyl)azo]-benzenesulfonamide (HPABS) was successfully studied by resonance Raman (rR) spectroscopy. HPABS is a synthetic competitive inhibitor (K(i)=7.17 x 10(-6)M) for the cresolase activity with a large extinction coefficient at 365 nm. Upon reacting with MT, HPABS produced an enzyme-inhibitor (EI) complex with sufficiently long life span. Analyzing the ensuing spectrum indicates that the azo tautomer of HPABS binds to the enzyme and retains its geometrical isomeric form in the EI complex. The observed changes in the rR spectrum of HPABS after binding to MT support the idea that an electrophilic attack on the inhibitor has happened. Similar experiments were designed for studying the oxidase activity of MT. However, the enzymatic reaction, even in the presence of 4-[(2,4-dinitrophenyl)azo]-1,2-benzenediols was still fast enough to tan the reaction solution quickly and render its rR spectrum impregnable background.