Reduction of alternative electron acceptors drives biofilm formation in Shewanella algae.

Shewanella spp. possess a broad respiratory versatility, which contributes to the occupation of hypoxic and anoxic environmental or host-associated niches. Here, we observe a strain-specific induction of biofilm formation in response to supplementation with the anaerobic electron acceptors dimethyl sulfoxide (DMSO) and nitrate in a panel of Shewanella algae isolates. The respiration-driven biofilm response is not observed in DMSO and nitrate reductase deletion mutants of the type strain S. algae CECT 5071, and can be restored upon complementation with the corresponding reductase operon(s) but not by an operon containing a catalytically inactive nitrate reductase. The distinct transcriptional changes, proportional to the effect of these compounds on biofilm formation, include cyclic di-GMP (c-di-GMP) turnover genes. In support, ectopic expression of the c-di-GMP phosphodiesterase YhjH of Salmonella Typhimurium but not its catalytically inactive variant decreased biofilm formation. The respiration-dependent biofilm response of S. algae may permit differential colonization of environmental or host niches.

Concentration Dependent Effect of Plant Root Exudates on the Chemosensory Systems of Pseudomonas putida KT2440.

Plant root colonization by rhizobacteria can protect plants against pathogens and promote plant growth, and chemotaxis to root exudates was shown to be an essential prerequisite for efficient root colonization. Since many chemoattractants control the transcript levels of their cognate chemoreceptor genes, we have studied here the transcript levels of the 27 Pseudomonas putida KT2440 chemoreceptor genes in the presence of different maize root exudate (MRE) concentrations. Transcript levels were increased for 10 chemoreceptor genes at low MRE concentrations, whereas almost all receptor genes showed lower transcript levels at high MRE concentrations. The exposure of KT2440 to different MRE concentrations did not alter c-di-GMP levels, indicating that changes in chemoreceptor transcripts are not mediated by this second messenger. Data suggest that rhizosphere colonization unfolds in a temporal fashion. Whereas at a distance to the root, exudates enhance chemoreceptor gene transcript levels promoting in turn chemotaxis, this process is reversed in root vicinity, where the necessity of chemotaxis toward the root may be less important. Insight into KT2440 signaling processes were obtained by analyzing mutants defective in the three cheA paralogous genes. Whereas a mutant in cheA1 showed reduced c-di-GMP levels and impaired biofilm formation, a cheA2 mutant was entirely deficient in MRE chemotaxis, indicating the existence of homologs of the P. aeruginosa wsp and che (chemotaxis) pathways. Signaling through both pathways was important for efficient maize root colonization. Future studies will show whether the MRE concentration dependent effect on chemoreceptor gene transcript levels is a feature shared by other species.

Riboswitches as Potential Targets for the Development of Anti-Biofilm Drugs.

In nature, bacteria can exist as single motile cells or as sessile cellular community, known as microbial biofilms. Bacteria within biofilms are embedded in a self-produced extracellular matrix that makes them more resistant to antibiotic treatment and responses of the host immune system. Microbial biofilms are very important in medicine since they are associated with several human diseases such as dental caries, periodontitis, otitis media, infective endocarditis, infectious kidney stones, osteomyelitis or prostatitis. In addition, biofilms formed on the surface of clinical devices such as pacemakers, implants and catheters are difficult to treat, which underlines the clinical relevance of biofilm formation. At the molecular level, the switch from the planktonic state to biofilm formation is regulated primarily by bis- (3'-5)-cyclic dimeric guanosine monophosphate (c-di-GMP). C-di-GMP performs its function by binding to a wide variety of proteins, but also to riboswitches. C-di-GMP riboswitches are RNA regulatory elements located in the 5'-untranslated regions (5'-UTRs) of RNA messengers (mRNA) from genes involved in virulence, motility and biofilm formation, which are regulated by changes in the intracellular concentration of c-di-GMP. This review discusses the role of c-di-GMP responsive riboswitches as potential targets for the design of anti-biofilm agents.

Identification of a Chemoreceptor in Pseudomonas aeruginosa That Specifically Mediates Chemotaxis Toward α-Ketoglutarate.

Pseudomonas aeruginosa is an ubiquitous pathogen able to infect humans, animals, and plants. Chemotaxis was found to be associated with the virulence of this and other pathogens. Although established as a model for chemotaxis research, the majority of the 26 P. aeruginosa chemoreceptors remain functionally un-annotated. We report here the identification of PA5072 (named McpK) as chemoreceptor for α-ketoglutarate (αKG). High-throughput thermal shift assays and isothermal titration calorimetry studies (ITC) of the recombinant McpK ligand binding domain (LBD) showed that it recognizes exclusively α-ketoglutarate. The ITC analysis indicated that the ligand bound with positive cooperativity (Kd1 = 301 µM, Kd2 = 81 µM). McpK is predicted to possess a helical bimodular (HBM) type of LBD and this and other studies suggest that this domain type may be associated with the recognition of organic acids. Analytical ultracentrifugation (AUC) studies revealed that McpK-LBD is present in monomer-dimer equilibrium. Alpha-KG binding stabilized the dimer and dimer self-dissociation constants of 55 µM and 5.9 µM were derived for ligand-free and αKG-bound forms of McpK-LBD, respectively. Ligand-induced LBD dimer stabilization has been observed for other HBM domain containing receptors and may correspond to a general mechanism of this protein family. Quantitative capillary chemotaxis assays demonstrated that P. aeruginosa showed chemotaxis to a broad range of αKG concentrations with maximal responses at 500 µM. Deletion of the mcpK gene reduced chemotaxis over the entire concentration range to close to background levels and wild type like chemotaxis was recovered following complementation. Real-time PCR studies indicated that the presence of αKG does not modulate mcpK expression. Since αKG is present in plant root exudates it was investigated whether the deletion of mcpK altered maize root colonization. However, no significant changes with respect to the wild type strain were observed. The existence of a chemoreceptor specific for αKG may be due to its central metabolic role as well as to its function as signaling molecule. This work expands the range of known chemoreceptor types and underlines the important physiological role of chemotaxis toward tricarboxylic acid cycle intermediates.

Correlation between signal input and output in PctA and PctB amino acid chemoreceptor of Pseudomonas aeruginosa.

The PctA and PctB chemoreceptors of Pseudomonas aeruginosa mediate chemotaxis toward amino acids. A general feature of signal transduction processes is that a signal input is converted into an output. We have generated chimeras combining the Tar signaling domain with either the PctA or PctB ligand binding domain (LBD). Escherichia coli harboring either PctA-Tar or PctB-Tar mediated chemotaxis toward amino acids. The responses of both chimeras were determined using fluorescence resonance energy transfer, and the derived EC50 values are a measure of output. PctA-Tar and PctB-Tar responded to 19 and 11 L-amino acids respectively. The EC50 values of PctA-Tar responses differed by more than three orders of magnitude, whereas PctB-Tar responded preferentially to L-Gln. The comparison of amino acid binding constants and the corresponding EC50 values for both receptors revealed statistically significant correlations between inputs and outputs. PctA and PctB possess a double PDC (PhoQ-DcuS-CitA) LBD - a family of binding domain found in various other amino acid chemoreceptors. Similarly, various chemoreceptors share the preferential response to certain amino acids (e.g. L-Cys, L-Ser and L-Thr) that we observed for PctA. Defining the specific inputs and outputs of these chemoreceptors is an important step toward better understanding of their physiological role.

Glucose conjugation of anti-HIV-1 oligonucleotides containing unmethylated CpG motifs reduces their immunostimulatory activity.

Antisense oligodeoxynucleotides (ODNs) are short synthetic DNA polymers complementary to a target RNA sequence. They are commonly designed to halt a biological event, such as translation or splicing. ODNs are potentially useful therapeutic agents for the treatment of different human diseases. Carbohydrate-ODN conjugates have been reported to improve the cell-specific delivery of ODNs through receptor mediated endocytosis. We tested the anti-HIV activity and biochemical properties of the 5'-end glucose-conjugated GEM 91 ODN targeting the initiation codon of the gag gene of HIV-1 RNA in cell-based assays. The conjugation of a glucose residue significantly reduces the immunostimulatory effect without diminishing its potent anti-HIV-1 activity. No significant effects were observed in either ODN stability in serum, in vitro degradation of antisense DNA-RNA hybrids by RNase H, cell toxicity, cellular uptake and ability to interfere with genomic HIV-1 dimerisation.

RecA protein plays a role in the chemotactic response and chemoreceptor clustering of Salmonella enterica.

The RecA protein is the main bacterial recombinase and the activator of the SOS system. In Escherichia coli and Salmonella enterica sv. Typhimurium, RecA is also essential for swarming, a flagellar-driven surface translocation mechanism widespread among bacteria. In this work, the direct interaction between RecA and the CheW coupling protein was confirmed, and the motility and chemotactic phenotype of a S. Typhimurium ΔrecA mutant was characterized through microfluidics, optical trapping, and quantitative capillary assays. The results demonstrate the tight association of RecA with the chemotaxis pathway and also its involvement in polar chemoreceptor cluster formation. RecA is therefore necessary for standard flagellar rotation switching, implying its essential role not only in swarming motility but also in the normal chemotactic response of S. Typhimurium.

Detection of immune response activation by exogenous nucleic acids by a multiplex RT-PCR method.

Transfection of mammalian cells or in vivo administration of nucleic acids can induce inflammatory cytokines and/or interferon response, which could significantly influence the ex vivo or in vivo applications of gene-targeting strategies based on nucleic acids. Further induction of the interferon and inflammatory related stress responses may result in off-target effects and toxicity. This work describes an original one-step multiplex reverse transcription polymerase chain reaction procedure, which allows testing the induction of interferon and proinflamatory related responses by nucleic acids in the cell system of choice. The developed procedure has been tested on mammalian cells transfected with ssRNA, dsRNA, enzymatically synthesized siRNA and synthetic oligodesoxyribonucleotides containing unmethylated cytosine-guanosine motifs. This procedure is a rapid and convenient screening assay that could be used routinely in both the clinical and the research laboratory to validate the stimulation of the immune system on mammalian cells by nucleic acids.

HIV RNA dimerisation interference by antisense oligonucleotides targeted to the 5' UTR structural elements.

The HIV-1 genome consists of two identical RNA molecules non-covalently linked by their 5' unstranslatable regions (5' UTR). The high level of sequence and structural conservation of this region correlates with its important functional involvement in the viral cycle, making it an attractive target for antiviral treatments based on antisense technology. Ten unmodified DNA antisense oligonucleotides (ODNs) targeted against different conserved structural elements within the 5' UTR were assayed for their capacity to interfere with HIV-1 RNA dimerisation, inhibit gene expression, and prevent virus production in cell cultures. The results show that, in addition to the well-characterised dimerisation initiation site (DIS), targeting of the AUG-containing structural element may reflect its direct role in HIV-1 genomic RNA dimerisation in vitro. Similarly, blocking the 3' end sequences of the stem-loop domain containing the primer biding site interferes with RNA dimerisation. Targeting the apical portion of the TAR element, however, appears to promote dimerisation. ODNs targeted against the conserved polyadenylation signal [Poly(A)], the primer binding site (PBS), the major splicing donor (SD) or the major packaging signal (Psi), and AUG-containing structural elements led to a highly efficient inhibition of HIV-1 gene expression and virus production in cell culture. Together, these results support the idea that ODNs possess great potential as molecular tools for the functional characterisation of viral RNA structural domains. Moreover, the targeting of these domains leads to the potent inhibition of viral replication, underscoring the potential of conserved structural RNA elements as antiviral targets.

Responses of Pseudomonas putida to toxic aromatic carbon sources.

A number of bacteria can use toxic compounds as carbon sources and have developed complex regulatory networks to protect themselves from the toxic effects of these compounds as well as to benefit from their nutritious properties. As a model system we have studied the responses of Pseudomonas putida strains to toluene. Although this compound is highly toxic, several strains are able to use it for growth. Particular emphasis was given to the responses in the context of taxis, resistance and toluene catabolism. P. putida strains analysed showed chemotactic movements towards toluene. Strain DOT-T1E was characterised by an extreme form of chemotaxis, termed hyperchemotaxis, which is mediated by the McpT chemoreceptor encoded by plasmid pGRT1. Close McpT homologs are found in a number of other plasmids encoding degradation pathways of toxic compounds. The pGRT1 plasmid harbours also the genes for the TtgGHI efflux pump which was identified as the primary determinant for the resistance of strain DOT-T1E towards toluene. Pump expression is controlled by the TtgV repressor in response to a wide range of different mono- and biaromatic compounds. Strain DOT-T1E is able to degrade toluene, benzene and ethylbenzene via the toluene dioxygenase (TOD) pathway. The expression of the pathway operon is controlled by the TodS/T two component system. The sensor kinase TodS recognizes toluene with nanomolar affinity, which in turn triggers an increase in its autophosphorylation and consequently transcriptional activation. Data suggest that transcriptional activation of the TOD pathway occurs at very low toluene concentrations whereas TtgV mediated induction of pump expression sets in as the toluene concentration further increases.

In vitro and ex vivo selection procedures for identifying potentially therapeutic DNA and RNA molecules.

It was only relatively recently discovered that nucleic acids participate in a variety of biological functions, besides the storage and transmission of genetic information. Quite apart from the nucleotide sequence, it is now clear that the structure of a nucleic acid plays an essential role in its functionality, enabling catalysis and specific binding reactions. In vitro selection and evolution strategies have been extremely useful in the analysis of functional RNA and DNA molecules, helping to expand our knowledge of their functional repertoire and to identify and optimize DNA and RNA molecules with potential therapeutic and diagnostic applications. The great progress made in this field has prompted the development of ex vivo methods for selecting functional nucleic acids in the cellular environment. This review summarizes the most important and most recent applications of in vitro and ex vivo selection strategies aimed at exploring the therapeutic potential of nucleic acids.

Inhibition of HIV-1 replication and dimerization interference by dual inhibitory RNAs.

The 5'-untranslated region (5'UTR) of the HIV-1 RNA is an attractive target for engineered ribozymes due to its high sequence and structural conservation. This region encodes several conserved structural RNA domains essential in key processes of the viral replication and infection cycles. This paper reports the inhibitory effects of catalytic antisense RNAs composed of two inhibitory RNA domains: an engineered ribozyme targeting the 5' UTR and a decoy or antisense domain of the dimerization initiation site (DIS). These chimeric molecules are able to cleave the HIV-1 5'UTR efficiently and prevent viral genome dimerization in vitro. Furthermore, catalytic antisense RNAs inhibited viral production up to 90% measured as p24 antigen levels in ex vivo assays. The use of chimeric RNA molecules targeting different domains represents an attractive antiviral strategy to be explored for the prevention of side effects from current drugs and of the rapid emergence of escape variants of HIV-1.

Inhibition of HIV-1 replication by RNA-based strategies.

The major etiologic agent of the acquired immunodeficiency syndrome (AIDS) is the human immunodeficiency virus type 1 (HIV-1), which belongs to the family of human retroviruses. This pandemic infection affects millions of people worldwide. The most efficient current treatment regimen for HIV-infected individuals combines two or more drugs targeting different HIV-specific enzymes. However, the emergence of multiple drug-resistant HIV-1 strains and the side effects of drug-based therapies make alternative approaches for the treatment of HIV infection and AIDS necessary. RNA-based antiviral approaches are among the most promising for developing long-term anti-HIV therapies. Anti-HIV-1 RNA-based strategies include ribozymes, antisense RNAs, RNA aptamers, RNA decoys, external guide sequences (EGS) for site-specific cleavage of RNA molecules with human ribonuclease P (RNase P), modified small nuclear RNA (RNAu) and small interfering RNAs (siRNAs). This review describes the main features and functions of viral and cellular targets as well as the different classes of RNA molecules that have been explored in developing therapeutic strategies against HIV infection. Many RNA-based strategies are already being tested in human clinical trials or are currently being developed for future trials.

Characterization of the first VanB vancomycin-resistant Enterococcus faecium isolated in a Spanish hospital.

We report the detection and characterization of the first vancomycin-resistant VanB-type Enterococcus faecium to be isolated in a Spanish hospital. Sequence analysis of the vanB gene showed that this isolate belonged to subtype vanB2. Moreover, PCR amplification analysis indicated that the vanB gene cluster was linked to a Tn5382-like transposon.