Staff member

Eduard Torrents Serra

Group Leader
Bacterial Infections: Antimicrobial Therapies
+34 934 034 756
CV Summary

Eduard Torrents obtained his PhD in microbiology in 2001 from the Science Faculty at the University Autonomous of Barcelona. During this time he spend 20 months at Karolinska Institute in Sweden with the supervision of Prof. Peter Reichard. From 2001 and 2003 he was a researcher at the R&D department at Biokit. S.A, an international company leader in clinical diagnostic reagents belonging to the Werfen group. He then moved in the laboratory of Prof Britt-Marie Sjöberg from the Department of Molecular Biology and Functional Genomics at the Stockholm University in Sweden first as a postdoc and then as a researcher (2004-2007). After his Postdoc he received a Ramon y Cajal tenure-track contract (2007) from the Spanish Ministry of Science to return to Spain at the Institute for Bioengineering of Catalonia (IBEC). In 2012, he obtained a position of Junior Group Leader at the (IBEC). His current research interests focus on the identification of new antimicrobial therapies and unravel the molecular mechanism underlying the transcripcional regulation of bacterial ribonucleotide reductase genes.

Staff member publications

Moya-Andérico, Laura, Admella, Joana, Torrents, Eduard, (2021). A clearing protocol for Galleria mellonella larvae: Visualization of internalized fluorescent nanoparticles New Biotechnology 60, 20-26

Light scattering is a challenge for imaging three-dimensional organisms. A number of new tissue clearing methodologies have been described in recent years, increasing the utilities of clearing techniques to obtain transparent samples. Here, we describe the optimization of a suitable and novel protocol for clearing Galleria mellonella larvae, an alternative infection animal model with a promising potential for the toxicological evaluation of different molecules and materials. This has allowed the visualization of internalised fluorescent nanoparticles using confocal microscopy, opening the door to a wide range of different applications.

Keywords: Nanotoxicology, Tissue clearance

Blanco-Cabra, Núria, Paetzold, Bernhard, Ferrar, Tony, Mazzolini, Rocco, Torrents, Eduard, Serrano, Luis, Lluch-Senar, Maria, (2020). Characterization of different alginate lyases for dissolving Pseudomonas aeruginosa biofilms Scientific Reports 10, (1), 9390

Aggregates of Pseudomonas aeruginosa form a protective barrier against antibiotics and the immune system. These barriers, known as biofilms, are associated with several infectious diseases. One of the main components of these biofilms is alginate, a homo- and hetero-polysaccharide that consists of β-D-mannuronate (M) and α-L-guluronate (G) units. Alginate lyases degrade this sugar and have been proposed as biotherapeutic agents to dissolve P. aeruginosa biofilms. However, there are contradictory reports in the literature regarding the efficacy of alginate lyases against biofilms and their synergistic effect with antibiotics. We found that most positive reports used a commercial crude extract from Flavobacterium multivorum as the alginate lyase source. By using anion exchange chromatography coupled to nano LC MS/MS, we identified two distinct enzymes in this extract, one has both polyM and polyG (polyM/G) degradation activities and it is similar in sequence to a broad-spectrum alginate lyase from Flavobacterium sp. S20 (Alg2A). The other enzyme has only polyG activity and it is similar in sequence to AlyA1 from Zobellia galactanivorans. By characterizing both of these enzymes together with three recombinant alginate lyases (a polyM, a polyG and a polyM/G), we showed that only enzymes with polyM/G activity such as Alg2A and A1-II’ (alginate lyase from Sphingomonas sp.) are effective in dissolving biofilms. Furthermore, both activities are required to have a synergistic effect with antibiotics.

Del Mar Cendra, Maria, Torrents, Eduard, (2020). Differential adaptability between reference strains and clinical isolates of Pseudomonas aeruginosa into the lung epithelium intracellular lifestyle Virulence 11, (1), 862-876

Intracellular invasion is an advantageous mechanism used by pathogens to evade host defense and antimicrobial therapy. In patients, the intracellular microbial lifestyle can lead to infection persistence and recurrence, thus worsening outcomes. Lung infections caused by Pseudomonas aeruginosa, especially in cystic fibrosis (CF) patients, are often aggravated by intracellular invasion and persistence of the pathogen. Proliferation of the infectious species relies on a continuous deoxyribonucleotide (dNTP) supply, for which the ribonucleotide reductase enzyme (RNR) is the unique provider. The large genome plasticity of P. aeruginosa and its ability to rapidly adapt to different environments are challenges for studying the pathophysiology associated with this type of infection. Using different reference strains and clinical isolates of P. aeruginosa independently combined with alveolar (A549) and bronchial (16HBE14o- and CF-CFBE41o-) epithelial cells, we analyzed host–pathogen interactions and intracellular bacterial persistence with the aim of determining a cell type-directed infection promoted by the P. aeruginosa strains. The oscillations in cellular toxicity and oxygen consumption promoted by the intracellular persistence of the strains were also analyzed among the different infectious lung models. Significantly, we identified class II RNR as the enzyme that supplies dNTPs to intracellular P. aeruginosa. This discovery could contribute to the development of RNR-targeted strategies against the chronicity occurring in this type of lung infection. Overall our study demonstrates that the choice of bacterial strain is critical to properly study the type of infectious process with relevant translational outcomes.

Keywords: Pseudomonas aeruginosa, Intracellular persistence, Lung, Epithelial cells, Clinical isolates, Host-pathogen interactions, Intracellular lifestyle, Chronic infections, Cystic fibrosis, Ribonucleotide reductase

Pedraz, Lucas, Blanco-Cabra, Núria, Torrents, Eduard, (2020). Gradual adaptation of facultative anaerobic pathogens to microaerobic and anaerobic conditions The FASEB Journal 34, (2), 2912-2928

Many notable human pathogens are facultative anaerobes. These pathogens exhibit redundant metabolic pathways and a whole array of regulatory systems to adapt to changing oxygen levels. However, our knowledge of facultative anaerobic pathogens is mostly based on fully aerobic or anaerobic cultures, which does not reflect real infection conditions, while the microaerobic range remains understudied. Here, we examine the behavior of pathogenic and nonpathogenic strains of two facultative anaerobes, Escherichia coli and Pseudomonas aeruginosa, during the aerobic-anaerobic transition. To do so, we introduce a new technique named AnaeroTrans, in which we allow self-consumption of oxygen by steady-state cultures and monitor the system by measuring the gas-phase oxygen concentration. We explore the different behavior of the studied species toward oxygen and examine how this behavior is associated with the targeted infection sites. As a model, we characterize the adaptation profile of the ribonucleotide reductase network, a complex oxygen-dependent enzymatic system responsible for the generation of the deoxyribonucleotides. We also explore the actions of the most important anaerobic regulators and how these regulators influence bacterial fitness. Our results allow us to classify the different elements that compose the aerobic-anaerobic transition into reproducible stages, thus showing the different adaptation mechanisms of the studied species.

Moya-Andérico, Laura, Admella, Joana, Fernandes, Rodrigo, Torrents, Eduard, (2020). Monitoring Gene Expression during a Galleria mellonella Bacterial Infection Microorganisms 8, (11), 1798

Galleria mellonella larvae are an alternative in vivo model that has been extensively used to study the virulence and pathogenicity of different bacteria due to its practicality and lack of ethical constraints. However, the larvae possess intrinsic autofluorescence that obstructs the use of fluorescent proteins to study bacterial infections, hence better methodologies are needed. Here, we report the construction of a promoter probe vector with bioluminescence expression as well as the optimization of a total bacterial RNA extraction protocol to enhance the monitoring of in vivo infections. By employing the vector to construct different gene promoter fusions, variable gene expression levels were efficiently measured in G. mellonella larvae at various time points during the course of infection and without much manipulation of the larvae. Additionally, our optimized RNA extraction protocol facilitates the study of transcriptional gene levels during an in vivo infection. The proposed methodologies will greatly benefit bacterial infection studies as they can contribute to a better understanding of the in vivo infection processes and pathogen–mammalian host interactions.

Keywords: Galleria mellonella, P. aeruginosa, Hemolymph, Hemocytes, Bioluminescence, Promoter probe vector, Optimized RNA extraction, Ribonucleotide reductases

Bach-Griera, Marc, Campo-Pérez, Víctor, Barbosa, Sandra, Traserra, Sara, Guallar-Garrido, Sandra, Moya-Andérico, Laura, Herrero-Abadía, Paula, Luquin, Marina, Rabanal, Rosa Maria, Torrents, Eduard, Julián, Esther, (2020). Mycolicibacterium brumae is a safe and non-toxic immunomodulatory agent for cancer treatment Vaccines 8, (2), 198

Intravesical Mycobacterium bovis Bacillus Calmette–Guérin (BCG) immunotherapy remains the gold-standard treatment for non-muscle-invasive bladder cancer patients, even though half of the patients develop adverse events to this therapy. On exploring BCG-alternative therapies, Mycolicibacterium brumae, a nontuberculous mycobacterium, has shown outstanding anti-tumor and immunomodulatory capabilities. As no infections due to M. brumae in humans, animals, or plants have been described, the safety and/or toxicity of this mycobacterium have not been previously addressed. In the present study, an analysis was made of M. brumae- and BCG-intravenously-infected severe combined immunodeficient (SCID) mice, M. brumae-intravesically-treated BALB/c mice, and intrahemacoelic-infected-Galleria mellonella larvae. Organs from infected mice and the hemolymph from larvae were processed to count bacterial burden. Blood samples from mice were also taken, and a wide range of hematological and biochemical parameters were analyzed. Finally, histopathological alterations in mouse tissues were evaluated. Our results demonstrate the safety and non-toxic profile of M. brumae. Differences were observed in the biochemical, hematological and histopathological analysis between M. brumae and BCG-infected mice, as well as survival curves rates and colony forming units (CFU) counts in both animal models. M. brumae constitutes a safe therapeutic biological agent, overcoming the safety and toxicity disadvantages presented by BCG in both mice and G. mellonella animal models.

Keywords: Bladder cancer, Nontuberculous mycobacteria, BCG, Safety, Galleria mellonella, Mice

Rahman, Abdel, Ganesh, Swapnil, Torrents, Eduard, Jahan, Nusrat, Wedyan, Moh'd Ali, Qaisi, Ali M., Al-Tawaha, Abdelrazzaq, (2020). Algal viruses Applied Plant Virology (ed. Awasthi, L. P.), Academic Press (London, UK) Advances, Detection, and Antiviral Strategies, 237-246

In marine ecosystems, the algae community is abundant and plays an important role in the food web. On the other hand, algal viruses have different advantages and disadvantages depending on the properties, such as applications of algal viruses in the advancement of molecular biology and for enhancement of biofuel production. Many environmental factors affecting growth and development of algae and virus such as temperature, salinity, ultraviolet radiation, photosynthetic active radiation, nutrients, inorganic particles, organic particles, CO2 concentration, and pH.

Keywords: Algal viruses, Biofuel production, Nutrients

Checa, Marti, Millán, Rubén, Blanco, Núria, Torrents, Eduard, Fabregas, Rene, Gomila, Gabriel, (2019). Mapping the dielectric constant of a single bacterial cell at the nanoscale with scanning dielectric force volume microscopy Nanoscale 11, 20809-20819

Mapping the dielectric constant at the nanoscale of samples showing a complex topography, such as non-planar nanocomposite materials or single cells, poses formidable challenges to existing nanoscale dielectric microscopy techniques. Here we overcome these limitations by introducing Scanning Dielectric Force Volume Microscopy. This scanning probe microscopy technique is based on the acquisition of electrostatic force approach curves at every point of a sample and its post-processing and quantification by using a computational model that incorporates the actual measured sample topography. The technique provides quantitative nanoscale images of the local dielectric constant of the sample with unparalleled accuracy, spatial resolution and statistical significance, irrespectively of the complexity of its topography. We illustrate the potential of the technique by presenting a nanoscale dielectric constant map of a single bacterial cell, including its small-scale appendages. The bacterial cell shows three characteristic equivalent dielectric constant values, namely, εr,bac1=2.6±0.2, εr,bac2=3.6±0.4 and εr,bac3=4.9±0.5, which enable identifying different dielectric properties of the cell wall and of the cytoplasmatic region, as well as, the existence of variations in the dielectric constant along the bacterial cell wall itself. Scanning Dielectric Force Volume Microscopy is expected to have an important impact in Materials and Life Sciences where the mapping of the dielectric properties of samples showing complex nanoscale topographies is often needed.

Vukomanovic, M., Torrents, E., (2019). High time resolution and high signal-to-noise monitoring of the bacterial growth kinetics in the presence of plasmonic nanoparticles Journal of Nanobiotechnology 17, (1), 21

Background: Emerging concepts for designing innovative drugs (i.e., novel generations of antimicrobials) frequently include nanostructures, new materials, and nanoparticles (NPs). Along with numerous advantages, NPs bring limitations, partly because they can limit the analytical techniques used for their biological and in vivo validation. From that standpoint, designing innovative drug delivery systems requires advancements in the methods used for their testing and investigations. Considering the well-known ability of resazurin-based methods for rapid detection of bacterial metabolisms with very high sensitivity, in this work we report a novel optimization for tracking bacterial growth kinetics in the presence of NPs with specific characteristics, such as specific optical properties. Results: Arginine-functionalized gold composite (HAp/Au/arginine) NPs, used as the NP model for validation of the method, possess plasmonic properties and are characterized by intensive absorption in the UV/vis region with a surface plasmon resonance maximum at 540 nm. Due to the specific optical properties, the NP absorption intensively interferes with the light absorption measured during the evaluation of bacterial growth (optical density; OD600). The results confirm substantial nonspecific interference by NPs in the signal detected during a regular turbidity study used for tracking bacterial growth. Instead, during application of a resazurin-based method (Presto Blue), when a combination of absorption and fluorescence detection is applied, a substantial increase in the signal-to-noise ratio is obtained that leads to the improvement of the accuracy of the measurements as verified in three bacterial strains tested with different growth rates (E. coli, P. aeruginosa, and S. aureus). Conclusions: Here, we described a novel procedure that enables the kinetics of bacterial growth in the presence of NPs to be followed with high time resolution, high sensitivity, and without sampling during the kinetic study. We showed the applicability of the Presto Blue method for the case of HAp/Au/arginine NPs, which can be extended to various types of metallic NPs with similar characteristics. The method is a very easy, economical, and reliable option for testing NPs designed as novel antimicrobials.

Keywords: Antimicrobial nanoparticles, Arginine-functionalized gold, Bacterial growth kinetics, Plasmonic nanoparticles, Presto Blue

Ohui, K., Afanasenko, E., Bacher, F., Ting, R. L. X., Zafar, A., Blanco-Cabra, N., Torrents, E., Dömötör, O., May, N. V., Darvasiova, D., Enyedy, Éva A., Popovi, Reynisson, J., Rapta, P., Babak, M. V., Pastorin, G., Arion, V. B., (2019). New water-soluble copper(II) complexes with morpholine-thiosemicarbazone hybrids: Insights into the anticancer and antibacterial mode of action Journal of Medicinal Chemistry 62, (2), 512-530

Six morpholine-(iso)thiosemicarbazone hybrids HL1–HL6 and their Cu(II) complexes with good-to-moderate solubility and stability in water were synthesized and characterized. Cu(II) complexes [Cu(L1–6)Cl] (1–6) formed weak dimeric associates in the solid state, which did not remain intact in solution as evidenced by ESI-MS. The lead proligands and Cu(II) complexes displayed higher antiproliferative activity in cancer cells than triapine. In addition, complexes 2–5 were found to specifically inhibit the growth of Gram-positive bacteria Staphylococcus aureus with MIC50 values at 2–5 μg/mL. Insights into the processes controlling intracellular accumulation and mechanism of action were investigated for 2 and 5, including the role of ribonucleotide reductase (RNR) inhibition, endoplasmic reticulum stress induction, and regulation of other cancer signaling pathways. Their ability to moderately inhibit R2 RNR protein in the presence of dithiothreitol is likely related to Fe chelating properties of the proligands liberated upon reduction.

Blanco-Cabra, N., Vega-Granados, K., Moya-Andérico, L., Vukomanovic, M., Parra, A., Álvarez De Cienfuegos, L., Torrents, E., (2019). Novel oleanolic and maslinic acid derivatives as a promising treatment against Bacterial biofilm in nosocomial infections: An in vitro and in vivo study ACS Infectious Diseases 5, (9), 1581-1589

Oleanolic acid (OA) and maslinic acid (MA) are pentacyclic triterpenic compounds that abound in industrial olive oil waste. These compounds have renowned antimicrobial properties and lack cytotoxicity in eukaryotic cells as well as resistance mechanisms in bacteria. Despite these advantages, their antimicrobial activity has only been tested in vitro, and derivatives improving this activity have not been reported. In this work, a set of 14 OA and MA C-28 amide derivatives have been synthesized. Two of these derivatives, MA-HDA and OA-HDA, increase the in vitro antimicrobial activity of the parent compounds while reducing their toxicity in most of the Gram-positive bacteria tested, including a methicillin-resistant Staphylococcus aureus-MRSA. MA-HDA also shows an enhanced in vivo efficacy in a Galleria mellonella invertebrate animal model of infection. A preliminary attempt to elucidate their mechanism of action revealed that these compounds are able to penetrate and damage the bacterial cell membrane. More significantly, their capacity to reduce antibiofilm formation in catheters has also been demonstrated in two sets of conditions: a static and a more challenged continuous-flow S. aureus biofilm.

Keywords: Antibiofilm, Galleria mellonella, In vitro and in vivo antimicrobials, Maslinic and oleanolic acids, Natural products, Staphylococcus aureus

Cendra, Maria del Mar, Blanco-Cabra, Núria, Pedraz, Lucas, Torrents, Eduard, (2019). Optimal environmental and culture conditions allow the in vitro coexistence of Pseudomonas aeruginosa and Staphylococcus aureus in stable biofilms Scientific Reports 9, (1), 16284

The coexistence between species that occurs in some infections remains hard to achieve in vitro since bacterial fitness differences eventually lead to a single organism dominating the mixed culture. Pseudomonas aeruginosa and Staphylococcus aureus are major pathogens found growing together in biofilms in disease-affected lungs or wounds. Herein, we tested and analyzed different culture media, additives and environmental conditions to support P. aeruginosa and S. aureus coexistence in vitro. We have unraveled the potential of DMEM to support the growth of these two organisms in mature cocultured biofilms (three days old) in an environment that dampens the pH rise. Our conditions use equal initial inoculation ratios of both strains and allow the stable formation of separate S. aureus microcolonies that grow embedded in a P. aeruginosa biofilm, as well as S. aureus biofilm overgrowth when bovine serum albumin is added to the system. Remarkably, we also found that S. aureus survival is strictly dependent on a well-characterized phenomenon of oxygen stratification present in the coculture biofilm. An analysis of differential tolerance to gentamicin and ciprofloxacin treatment, depending on whether P. aeruginosa and S. aureus were growing in mono- or coculture biofilms, was used to validate our in vitro coculture conditions.

Keywords: Applied microbiology, Biofilms

Lozano, Helena, Fabregas, Rene, Blanco, Núria, Millán, Rubén, Torrents, Eduard, Fumagalli, Laura, Gomila, Gabriel, (2018). Dielectric constant of flagellin proteins measured by scanning dielectric microscopy Nanoscale 10, 19188-19194

The dielectric constant of flagellin proteins in flagellar bacterial filaments ~10-20 nm in diameter is measured using Scanning Dielectric Microscopy. We obtain for two different bacterial species (Shewanella oneidensis MR-1 and Pseudo-monas aeruginosa PAO1) similar relative dielectric constant values εSo = 4.3 ± 0.6 and εPa = 4.5 ± 0.7, respectively, despite their different structure and aminoacid sequence. Present results show the applicability of Scanning Dielectric Microscopy to nanoscale filamentous protein complexes, and to general 3D macromolecular protein geometries, thus opening new avenues to study the relationship between dielectric response and protein structure and function.

Basas, Jana, Palau, Marta, Ratia, Carlos, Luis Del Pozo, José, Martín-Gómez, María Teresa, Gomis, Xavier, Torrents, Eduard, Almirante, Benito, Gavaldà, Joan, (2018). High-dose daptomycin is effective as an antibiotic-lock therapy in a rabbit model of Staphylococcus epidermidis catheter-related infection Antimicrobial Agents and Chemotherapy 62, (2), e01777

Long-term catheter-related bloodstream infections (CRBSI) involving coagulase-negative Staphylococci are associated with poor patient outcomes, increased hospitalization and high treatment costs. The use of vancomycin-lock therapy has been an important step forward to treat these biofilms although failures appear in 20% of patients. In this study, we report that a high dose of daptomycin-lock therapy may offer a therapeutic advantage for these CRBSI in just 24 h of treatment.

Urrea, L., Segura, Miriam, Masuda-Suzukake, M., Hervera, A., Pedraz, L., Aznar, J. M. G., Vila, M., Samitier, J., Torrents, E., Ferrer, Isidro, Gavín, R., Hagesawa, M., Del Río, J. A., (2018). Involvement of cellular prion protein in α-synuclein transport in neurons Molecular Neurobiology 55, (3), 1847-1860

The cellular prion protein, encoded by the gene Prnp, has been reported to be a receptor of β-amyloid. Their interaction is mandatory for neurotoxic effects of β-amyloid oligomers. In this study, we aimed to explore whether the cellular prion protein participates in the spreading of α-synuclein. Results demonstrate that Prnp expression is not mandatory for α-synuclein spreading. However, although the pathological spreading of α-synuclein can take place in the absence of Prnp, α-synuclein expanded faster in PrPC-overexpressing mice.

Keywords: Amyloid spreading, Microfluidic devices, Prnp, Synuclein

Crespo, Anna, Blanco-Cabra, N., Torrents, Eduard, (2018). Aerobic vitamin B12 biosynthesis is essential for pseudomonas aeruginosa class II ribonucleotide reductase activity during planktonic and biofilm growth Frontiers in Microbiology 9, (986), Article 986

P. aeruginosa is a major pathogenic bacterium in chronic infections and is a model organism for studying biofilms. P. aeruginosa is considered an aerobic bacterium, but in the presence of nitrate, it also grows in anaerobic conditions. Oxygen diffusion through the biofilm generates metabolic and genetic diversity in P. aeruginosa growth, such as in ribonucleotide reductase activity. These essential enzymes are necessary for DNA synthesis and repair. Oxygen availability determines the activity of the three-ribonucleotide reductase (RNR) classes. Class II and III RNRs are active in the absence of oxygen; however, class II RNRs, which are important in P. aeruginosa biofilm growth, require a vitamin B12 cofactor for their enzymatic activity. In this work, we elucidated the conditions in which class II RNRs are active due to vitamin B12 concentration constraints (biosynthesis or environmental availability). We demonstrated that increased vitamin B12 levels during aerobic, stationary and biofilm growth activate class II RNR activity. We also established that the cobN gene is essentially responsible for B12 biosynthesis under planktonic and biofilm growth. Our results unravel the mechanisms of dNTP synthesis by P. aeruginosa during biofilm growth, which appear to depend on the bacterial strain (laboratory-type or clinical isolate).

Keywords: Vitamin B12, Adenosylcobalamin, Ribonucleotide Reductases, Pseudomonas aeruginosa, NrdJ, Bacterial growth, Biofilm,Anaerobiosis

Pujol, E., Blanco-Cabra, N., Julián, E., Leiva, R., Torrents, E., Vázquez, S., (2018). Pentafluorosulfanyl-containing triclocarban analogs with potent antimicrobial activity Molecules 23, (11), 2853

Concerns have been raised about the long-term accumulating effects of triclocarban, a polychlorinated diarylurea widely used as an antibacterial soap additive, in the environment and in human beings. Indeed, the Food and Drug Administration has recently banned it from personal care products. Herein, we report the synthesis, antibacterial activity and cytotoxicity of novel N,N′-diarylureas as triclocarban analogs, designed by reducing one or more chlorine atoms of the former and/or replacing them by the novel pentafluorosulfanyl group, a new bioisostere of the trifluoromethyl group, with growing importance in drug discovery. Interestingly, some of these pentafluorosulfanyl-bearing ureas exhibited high potency, broad spectrum of antimicrobial activity against Gram-positive bacterial pathogens, and high selectivity index, while displaying a lower spontaneous mutation frequency than triclocarban. Some lines of evidence suggest a bactericidal mode of action for this family of compounds.

Keywords: Antibacterial, Gram-positive, N,N'-diarylureas, Pentafluorosulfanyl, Staphylococcus aureus, Triclocarban

Miret-Casals, L., Baelo, A., Julián, E., Astola, J., Lobo-Ruiz, A., Albericio, F., Torrents, E., (2018). Hydroxylamine derivatives as a new paradigm in the search of antibacterial agents ACS Omega 3, (12), 17057-17069

Serious infections caused by bacteria that are resistant to commonly used antibiotics have become a major global healthcare problem in the 21st century. Multidrug-resistant bacteria causing severe infections mainly grow in complex bacterial communities known as biofilms, in which bacterial resistance to antibacterial agents and to the host immune system is strengthened. As drug resistance is becoming a threatening problem, it is necessary to develop new antimicrobial agents with novel mechanisms of action. Here, we designed and synthesized a small library of N-substituted hydroxylamine (N-HA) compounds with antibacterial activity. These compounds, acting as radical scavengers, inhibit the bacterial ribonucleotide reductase (RNR) enzyme. RNR enzyme is essential for bacterial proliferation during infection, as it provides the building blocks for DNA synthesis and repair. We demonstrate the broad antimicrobial effect of several drug candidates against a variety of Gram-positive and Gram-negative bacteria, together with low toxicity toward eukaryotic cells. Furthermore, the most promising compounds can reduce the biomass of an established biofilm on Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli. This study settles the starting point to develop new N-hydroxylamine compounds as potential effective antibacterial agents to fight against drug-resistant pathogenic bacteria.

Crespo, Anna, Pedraz, Lucas, Van Der Hofstadt, Marc, Gomila, Gabriel, Torrents, Eduard, (2017). Regulation of ribonucleotide synthesis by the Pseudomonas aeruginosa two-component system AlgR in response to oxidative stress Scientific Reports 7, (1), 17892

Ribonucleotide reductases (RNR) catalyze the last step of deoxyribonucleotide synthesis, and are therefore essential to DNA-based life. Three forms of RNR exist: classes I, II, and III. While eukaryotic cells use only class Ia RNR, bacteria can harbor any combination of classes, granting them adaptability. The opportunistic pathogen Pseudomonas aeruginosa surprisingly encodes all three classes, allowing it to thrive in different environments. Here we study an aspect of the complex RNR regulation whose molecular mechanism has never been elucidated, the well-described induction through oxidative stress, and link it to the AlgZR two-component system, the primary regulator of the mucoid phenotype. Through bioinformatics, we identify AlgR binding locations in RNR promoters, which we characterize functionally through EMSA and physically through AFM imaging. Gene reporter assays in different growth models are used to study the AlgZR-mediated control on the RNR network under various environmental conditions and physiological states. Thereby, we show that the two-component system AlgZR, which is crucial for bacterial conversion to the mucoid phenotype associated with chronic disease, controls the RNR network and directs how the DNA synthesis pathway is modulated in mucoid and non-mucoid biofilms, allowing it to respond to oxidative stress.

Keywords: Bacterial genes, Bacteriology, Pathogens

Crespo, A., Gavaldà, J., Julián, E., Torrents, E., (2017). A single point mutation in class III ribonucleotide reductase promoter renders Pseudomonas aeruginosa PAO1 inefficient for anaerobic growth and infection Scientific Reports 7, (1), 13350

Pseudomonas aeruginosa strain PAO1 has become the reference strain in many laboratories. One enzyme that is essential for its cell division is the ribonucleotide reductase (RNR) enzyme that supplies the deoxynucleotides required for DNA synthesis and repair. P. aeruginosa is one of the few microorganisms that encodes three different RNR classes (Ia, II and III) in its genome, enabling it to grow and adapt to diverse environmental conditions, including during infection. In this work, we demonstrate that a lack of RNR activity induces cell elongation in P. aeruginosa PAO1. Moreover, RNR gene expression during anaerobiosis differs among P. aeruginosa strains, with class III highly expressed in P. aeruginosa clinical isolates relative to the laboratory P. aeruginosa PAO1 strain. A single point mutation was identified in the P. aeruginosa PAO1 strain class III RNR promoter region that disrupts its anaerobic transcription by the Dnr regulator. An engineered strain that induces the class III RNR expression allows P. aeruginosa PAO1 anaerobic growth and increases its virulence to resemble that of clinical strains. Our results demonstrate that P. aeruginosa PAO1 is adapted to laboratory conditions and is not the best reference strain for anaerobic or infection studies.

Keywords: Bacterial genes, Cellular microbiology, Pathogens

Morer, A., Basas, J., Colominas, S., Ratia, C., Gomis, X., Abellà, J., Torrents, E., Larrosa, N., Almirante, B., Gavaldà, J., (2017). Actividad de la electrólisis con corriente eléctrica directa continua de bajo amperaje en infecciones por biopelículas y por microorganismos XDR Gram-negativos Enfermedades Infecciosas y Microbiología Clínica XXI Congreso de la Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica (SEIMC) , Elsevier (Málaga, Spain) 35, (Espec. Cong. 1), 41

Necesitamos nuevas estrategias para combatir la resistencia a los antibióticos. Las infecciones crónicas asociadas con dispositivos médicos se asocian con una morbimortalidad significativa. Nuestro objetivo fue desarrollar una nueva estrategia frente a infecciones productoras de biopelículas y aquellas producidas por microorganismos gramnegativos XDR mediante el uso de una corriente eléctrica directa continua de bajo amperaje (CD). Esta estrategia se evaluó en presencia de suero fisiológico como electrolito para aproximarse a la situación in vivo.

Basas, J., Morer, A., Ratia, C., Rojo, E., Larrosa, N., Oliver, A., Cantón, R., Ferrer, R., Gomis, X., Grau, S., Vima, J., Torrents, E., Almirante, B., Gavaldà, J., (2017). Eficacia in vitro e in vivo de distintas combinaciones antibióticas para el tratamiento nebulizado de neumonía aguda frente a clones de alto riesgo de Pseudomonas aeruginosa. Enfermedades Infecciosas y Microbiología Clínica Enfermedades Infecciosas y Microbiología Clínica XXI Congreso de la Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica (SEIMC) , Elsevier (Málaga, Spain) 35, (Espec. Cong. 1), 126

El tratamiento de las infecciones respiratorias por cepas extremadamente resistentes (XDR) de P. aeruginosa (sólo sensibles a colistina/amikacina) es complicado, en ocasiones ineficaz y/o nefrotóxico. La nebulización con antibióticos parece una estrategia terapéutica adecuada frente a ese tipo de infecciones.

D'Auria, Giuseppe, Torrents, Eduard, Luquin, Marina, Comas, Iñaki, Julián, Esther, (2016). Draft genome sequence of Mycobacterium brumae ATCC 51384 Genome Announcements , 4, (2), e00237-16

Here, we report the draft genome sequence of Mycobacterium brumae type strain ATCC 51384. This is the first draft genome sequence of M. brumae, a nonpathogenic, rapidly growing, nonchromogenic mycobacterium, with immunotherapeutic capacities.

Noguera-Ortega, Estela, Secanella-Fandos, Silvia, Eraña, Hasier, Gasión, Jofre, Rabanal, Rosa M., Luquin, Marina, Torrents, Eduard, Julián, Esther, (2016). Nonpathogenic Mycobacterium brumae inhibits bladder cancer growth in vitro, ex vivo, and in vivo European Urology Focus , 2, (1), 67-76

Background Bacillus Calmette-Guérin (BCG) prevents tumour recurrence and progression in non–muscle-invasive bladder cancer (BC). However, common adverse events occur, including BCG infections. Objective To find a mycobacterium with similar or superior antitumour activity to BCG but with greater safety. Design In vitro, ex vivo, and in vivo comparisons of the antitumour efficacy of nonpathogenic mycobacteria and BCG. Intervention The in vitro antitumour activity of a broad set of mycobacteria was studied in seven different BC cell lines. The most efficacious was selected and its ex vivo capacity to activate immune cells and its in vivo antitumour activity in an orthotopic murine model of BC were investigated. Outcome measurements and statistical analysis Growth inhibition of BC cells was the primary outcome measurement. Parametric and nonparametric tests were use to analyse the in vitro results, and a Kaplan-Meier test was applied to measure survival in mycobacteria-treated tumour-bearing mice. Results and limitations Mycobacterium brumae is superior to BCG in inhibiting low-grade BC cell growth, and has similar effects to BCG against high-grade cells. M. brumae triggers an indirect antitumour response by activating macrophages and the cytotoxic activity of peripheral blood cells against BC cells. Although no significant differences were observed between BCG and M. brumae treatments in mice, M. brumae treatment prolonged survival in comparison to BCG treatment in tumour-bearing mice. In contrast to BCG, M. brumae does not persist intracellularly or in tumour-bearing mice, so the risk of infection is lower. Conclusions Our preclinical data suggest that M. brumae represents a safe and efficacious candidate as a therapeutic agent for non–muscle-invasive BC. Patient summary We investigated the antitumour activity of nonpathogenic mycobacteria in in vitro and in vivo models of non–muscle-invasive bladder cancer. We found that Mycobacterium brumae effectively inhibits bladder cancer growth and helps the host immune system to eradicate cancer cells, and is a promising agent for antitumour immunotherapy.

Keywords: Animal models, Bacillus Calmette-Guérin, Cytokines, Immunomodulation, Immunotherapy, Mycobacteria, Urothelial cell line

Noguera-Ortega, E., Rabanal, R. M., Secanella-Fandos, S., Torrents, E., Luquin, M., Julián, E., (2016). Gamma-irradiated mycobacteria enhance survival in bladder tumor bearing mice although less efficaciously than live mycobacteria Journal of Urology , 195, (1), 198-205

Purpose γ Irradiated Mycobacterium bovis bacillus Calmette-Guérin has shown in vitro and ex vivo antitumor activity. However, to our knowledge the potential antitumor capacity has not been demonstrated in vivo. We studied the in vivo potential of γ irradiated bacillus Calmette-Guérin and γ irradiated M. brumae, a saprophytic mycobacterium that was recently described as an immunotherapeutic agent. Materials and Methods The antitumor capacity of γ irradiated M. brumae was first investigated by analyzing the in vitro inhibition of bladder tumor cell proliferation and the ex vivo cytotoxic effect of M. brumae activated peripheral blood cells. The effect of γ irradiated M. brumae or bacillus Calmette-Guérin intravesical treatment was then compared to treatment with live mycobacteria in the orthotopic murine model of bladder cancer. Results Nonviable M. brumae showed a capacity to inhibit in vitro bladder cancer cell lines similar to that of live mycobacteria. However, its capacity to induce cytokine production was decreased compared to that of live M. brumae. γ Irradiated M. brumae could activate immune cells to inhibit tumor cell growth, although to a lesser extent than live mycobacteria. Finally, intravesical treatment with γ irradiated M. brumae or bacillus Calmette-Guérin significantly increased survival with respect to that of nontreated tumor bearing mice. Both γ irradiated mycobacteria showed lower survival rates than those of live mycobacteria but the minor efficacy of γ irradiated vs live mycobacteria was only significant for bacillus Calmette-Guérin. Conclusions Our results show that although γ irradiated mycobacteria is less efficacious than live mycobacteria, it induces an antitumor effect in vivo, avoiding the possibility of further mycobacterial infections.

Keywords: BCG vaccine, Gamma rays, Immunotherapy, Mycobacterium, Urinary bladder neoplasms

Basas, J., Morer, A., Ratia, C., Martín, M.T., del Pozo, J.L., Gomis, X., Rojo-Molinero, E., Torrents, E., Almirante, B., Gavaldà, J., (2016). Efficacy of anidulafungin in the treatment of experimental Candida parapsolosis catheter infection Journal of Antimicrobial Chemotherapy , 71, (10), 2895-2901

Objectives The effectiveness of anidulafungin versus liposomal amphotericin B (LAmB) for treating experimental Candida parapsilosis catheter-related infection by an antifungal-lock technique was assessed. Methods Two clinical strains of C. parapsilosis (CP12 and CP54) were studied. In vitro studies were used to determine the biofilm MICs (MBIC50 and MBIC90) by XTT reduction assay and LIVE/DEAD biofilm viability for anidulafungin and LAmB on 96-well microtitre polystyrene plates and silicone discs. An intravenous catheter was implanted in New Zealand white rabbits. Infection was induced by locking the catheter for 48 h with the inoculum. The 48 h antifungal-lock treatment groups included control, 3.3 mg/mL anidulafungin and 5.5 mg/mL LAmB. Results Anidulafungin showed better in vitro activity than LAmB against C. parapsilosis growing in biofilm on silicone discs. MBIC90 of LAmB: CP12, >1024 mg/L; CP54, >1024 mg/L. MBIC90 of anidulafungin: CP12, 1 mg/L; CP54, 1 mg/L (P ≤ 0.05). Moreover, only anidulafungin (1 mg/L) showed >90% non-viable cells in the LIVE/DEAD biofilm viability assay on silicone discs. No differences were observed between the in vitro susceptibility of anidulafungin or LAmB when 96-well plates were used. Anidulafungin achieved significant reductions relative to LAmB in log10 cfu recovered from the catheter tips for both strains (P ≤ 0.05). Only anidulafungin achieved negative catheter tip cultures (CP12 63%, CP54 73%, P ≤ 0.05). Conclusions Silicone discs may be a more reliable substrate for the study of in vitro biofilm susceptibility of C. parapsilosis. Anidulafungin-lock therapy showed the highest activity for experimental catheter-related infection with C. parapsilosis.

Noguera-Ortega, E., Blanco-Cabra, N., Rabanal, R.M., Sanchez-Chardi, A., Roldán, M., Torrents, E., Luquin, M., Julián, E., (2016). Mycobacteria emulsified in olive oil-in-water trigger a robust immune response in bladder cancer treatment Scientific Reports 6, 27232

The hydrophobic composition of mycobacterial cell walls leads to the formation of clumps when attempting to resuspend mycobacteria in aqueous solutions. Such aggregation may interfere in the mycobacteria-host cells interaction and, consequently, influence their antitumor effect. To improve the immunotherapeutic activity of Mycobacterium brumae, we designed different emulsions and demonstrated their efficacy. The best formulation was initially selected based on homogeneity and stability. Both olive oil (OO)- and mineral oil-in-water emulsions better preserved the mycobacteria viability and provided higher disaggregation rates compared to the others. But, among both emulsions, the OO emulsion increased the mycobacteria capacity to induce cytokines’ production in bladder tumor cell cultures. The OO-mycobacteria emulsion properties: less hydrophobic, lower pH, more neutralized zeta potential, and increased affinity to fibronectin than non-emulsified mycobacteria, indicated favorable conditions for reaching the bladder epithelium in vivo. Finally, intravesical OO-M. brumae-treated mice showed a significantly higher systemic immune response, together with a trend toward increased tumor-bearing mouse survival rates compared to the rest of the treated mice. The physicochemical characteristics and the induction of a robust immune response in vitro and in vivo highlight the potential of the OO emulsion as a good delivery vehicle for the mycobacterial treatment of bladder cancer.

Crespo, Anna, Pedraz, Lucas, Astola, Josep, Torrents, Eduard, (2016). Pseudomonas aeruginosa exhibits deficient biofilm formation in the absence of class II and III ribonucleotide reductases due to hindered anaerobic growth Frontiers in Microbiology 7, Article 688

Chronic lung infections by the ubiquitous and extremely adaptable opportunistic pathogen Pseudomonas aeruginosa correlate with the formation of a biofilm, where bacteria grow in association with an extracellular matrix and display a wide range of changes in gene expression and metabolism. This leads to increased resistance to physical stress and antibiotic therapies, while enhancing cell-to-cell communication. Oxygen diffusion through the complex biofilm structure generates an oxygen concentration gradient, leading to the appearance of anaerobic microenvironments. Ribonucleotide reductases (RNRs) are a family of highly sophisticated enzymes responsible for the synthesis of the deoxyribonucleotides, and they constitute the only de novo pathway for the formation of the building blocks needed for DNA synthesis and repair. P. aeruginosa is one of the few bacteria encoding all three known RNR classes (Ia, II, and III). Class Ia RNRs are oxygen dependent, class II are oxygen independent, and class III are oxygen sensitive. A tight control of RNR activity is essential for anaerobic growth and therefore for biofilm development. In this work we explored the role of the different RNR classes in biofilm formation under aerobic and anaerobic initial conditions and using static and continuous-flow biofilm models. We demonstrated the importance of class II and III RNR for proper cell division in biofilm development and maturation. We also determined that these classes are transcriptionally induced during biofilm formation and under anaerobic conditions. The molecular mechanism of their anaerobic regulation was also studied, finding that the Anr/Dnr system is responsible for class II RNR induction. These data can be integrated with previous knowledge about biofilms in a model where these structures are understood as a set of layers determined by oxygen concentration and contain cells with different RNR expression profiles, bringing us a step closer to the understanding of this complex growth pattern, essential for P. aeruginosa chronic infections.

Keywords: Pseudomonas aeruginosa, Ribonucleotide Reductases, Vitamin B 12, Anaerobic metabolism, Biofilm formation, DNA Synthesis, Oxygen diffusion, nrd genes.

Baelo, Aida, Julián, Esther, Torrents, Eduard, (2016). Methyl-hydroxylamine specifically inhibits ribonucleotide reductase activity in pathogenic bacteria New Biotechnology Biotech Annual Congress (BAC 2015) , Elsevier (Salamanca, Spain) 33, (3), 417

Infectious diseases constitute a tenacious and major public-health problem all over the world; the emergence and increasing prevalence of multi-drug resistant bacteria demand the discovery of new therapeutic approaches. Bacterial DNA synthesis opens new horizons in the discovery of new antibacterial targets due to remarkable differences to the eukaryotic system. During the course of an infection, a great number of bacteria need to multiply inside the body and, for that, active DNA synthesis with a balanced supply of deoxyribonucleotides (dNTPs) is required. RiboNucleotide Reductase (RNR) is the key enzyme that provides the nucleotide precursors for DNA replication and repair. This enzyme is a suitable target candidate for bacterial growth inhibition. In this work we have firstly identified the radical scavenger methyl-hydroxylamine (M-HA) as an efficacious antimicrobial agent that inhibits gram-negative and gram-positive pathogenic bacteria, targeting the RNR enzyme. Later, we have focused our work studying the ability of M-HA to inhibit the intracellular growth of Mycobacteria in macrophages, and the formation of Pseudomonas aeruginosa biofilms.

Pedraz, Lucas, Crespo, Anna, Torrents, Eduard, (2016). A single transcription factor behind all bacterial dNTP synthesis revealed as a novel antimicrobial target New Biotechnology Biotech Annual Congress (BAC 2015) , Elsevier (Salamanca, Spain) 33, (3), 410

Nowadays, the fear of infectious diseases is again increasing. Antibiotic-resistant bacterial strains are appearing worldwide, and so there is an urgent need to develop new antimicrobial drugs. Ribonucleotide Reductases (RNRs) are essential enzymes that catalyse the reduction of ribonucleotides (NTPs) to their corresponding deoxyribonucleotides (dNTPs), thereby forming the building blocks for DNA synthesis and repair. A drug able to inhibit bacterial Ribonucleotide Reductase activity would completely inhibit bacterial growth. Behind bacterial Ribonucleotide Reductase activity there is a complex regulon; although eukaryotic cells codify only for one RNR enzyme, bacteria can use three different RNR classes, granting them a huge adaptability. Pseudomonas aeruginosa is a major human opportunistic pathogen, causing severe lung chronic infections in cystic fibrosis and COPD patients. It codifies for all three RNR classes, in a complex regulon necessary for its adaptability and virulence. The main focus of this work is a transcription factor, called NrdR, which is present in almost all bacterial species, and completely absent in eukaryotic organisms. This factor acts as a central regulator of all RNR enzymes in bacteria, hence being behind all dNTP synthesis. We have studied how NrdR regulates RNR activity in P. aeruginosa, being able to this point to propose a first model of the NrdR regulon, and being a step closer to new antimicrobial therapies.

Julián, E., Rabanal, R. M., Secanella-Fandos, S., Torrents, E., Luquin, M., Noguera-Ortega, E., (2016). Eficacia de micobacterias Gamma-irradiadas en el tratamiento de cancer vesical no-músculo invasivo Enfermedades Infecciosas y Microbiología Clínica XX Congreso de la Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica (SEIMC) , Elsevier (Barcelona, Spain) 34, (SE1), 229

Las micobacterias son las únicas bacterias utilizadas en el tratamiento del cáncer. Concretamente Mycobacterium bovis BCG se instila intravesicalmente en pacientes de cáncer vesical no-músculo invasivo, tras la resección del tumor, con el fin de evitar recidivas. A pesar de su eficacia, BCG presenta numerosos efectos adversos, entre ellos casos de infección por BCG. En nuestro laboratorio hemos desarrollado dos estrategias para evitar el riesgo de infección. Por un lado, hemos mostrado la capacidad antitumoral in vitro de BCG muerta mediante gamma-irradiación. Por otro lado, hemos demostrado la capacidad antitumoral de Mycobacterium brumae viva, tanto en modelos in vitro como en el modelo animal de la enfermedad. Aun así, se desconoce el potencial antitumoral de estas micobacterias irradiadas in vivo, y si su eficacia es comparable. El objetivo fue evaluar la capacidad antitumoral de BCG y M. brumae gamma-irradiadas en el modelo murino de cáncer vesical.

Torrents, E., Baelo, Aida, Levato, R., Julián, E., Crespo, Anna, Astola, Josep, Gavaldà, J., Engel, E., Mateos-Timoneda, M.A., (2016). Mejora en la administración antibiotic para el tratamiento de infecciones en forma de biofilm con el uso de nanopartículas que disgregan la matriz extracellular Enfermedades Infecciosas y Microbiología Clínica XX Congreso de la Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica (SEIMC) , Elsevier (Barcelona, Spain) 34, (SE1), 31

Las infecciones causadas por bacterias formadoras de biopelículas o biofilms son una amenaza importante para los pacientes hospitalizados y suponen la principal causa de infecciones crónicas, como las producidas en la enfermedad pulmonar obstructiva crónica (EPOC) y la fibrosis quística. Existe una necesidad urgente de desarrollar nuevos antibióticos o nuevos enfoques terapéuticos que permitan el tratamiento de este tipo de infecciones ya que los antibióticos convencionales no logran eliminar las bacterias que están formando biofilms

Baelo, Aida, Levato, Riccardo, Julián, Esther, Crespo, Anna, Astola, José, Gavaldà, Joan, Engel, Elisabeth, Mateos-Timoneda, Miguel Angel, Torrents, Eduard, (2015). Disassembling bacterial extracellular matrix with DNase-coated nanoparticles to enhance antibiotic delivery in biofilm infections Journal of Controlled Release 209, 150-158

Abstract Infections caused by biofilm-forming bacteria are a major threat to hospitalized patients and the main cause of chronic obstructive pulmonary disease and cystic fibrosis. There is an urgent necessity for novel therapeutic approaches, since current antibiotic delivery fails to eliminate biofilm-protected bacteria. In this study, ciprofloxacin-loaded poly(lactic-co-glycolic acid) nanoparticles, which were functionalized with DNase I, were fabricated using a green-solvent based method and their antibiofilm activity was assessed against Pseudomonas aeruginosa biofilms. Such nanoparticles constitute a paradigm shift in biofilm treatment, since, besides releasing ciprofloxacin in a controlled fashion, they are able to target and disassemble the biofilm by degrading the extracellular DNA that stabilize the biofilm matrix. These carriers were compared with free-soluble ciprofloxacin, and ciprofloxacin encapsulated in untreated and poly(lysine)-coated nanoparticles. DNase I-activated nanoparticles were not only able to prevent biofilm formation from planktonic bacteria, but they also successfully reduced established biofilm mass, size and living cell density, as observed in a dynamic environment in a flow cell biofilm assay. Moreover, repeated administration over three days of DNase I-coated nanoparticles encapsulating ciprofloxacin was able to reduce by 95% and then eradicate more than 99.8% of established biofilm, outperforming all the other nanoparticle formulations and the free-drug tested in this study. These promising results, together with minimal cytotoxicity as tested on J774 macrophages, allow obtaining novel antimicrobial nanoparticles, as well as provide clues to design the next generation of drug delivery devices to treat persistent bacterial infections.

Keywords: Pseudomonas aeruginosa, Biofilm, Ciprofloxacin, DNase I, Nanoparticles

Dreux, Nicolas, Cendra, Maria del Mar, Massier, Sébastien, Darfeuille-Michaud, Arlette, Barnich, Nicolas, Torrents, Eduard, (2015). Ribonucleotide reductase NrdR as a novel regulator for motility and chemotaxis during adherent-invasive Escherichia coli infection Infection and Immunity , 83, (4), 1305-1317

A critical step in the life cycle of all organisms is the duplication of the genetic material during cell division. Ribonucleotide reductases (RNRs) are essential enzymes for this step because they control the de novo production of the deoxyribonucleotides required for DNA synthesis and repair. Enterobacteriaceae have three functional classes of RNRs (Ia, Ib and III), which are transcribed from separate operons and encoded, respectively by the genes nrdAB, nrdHIEF and nrdDG. Here, we investigated the role of RNRs in the virulence of adherent-invasive E. coli (AIEC) isolated from Crohn's disease (CD) patients. Interestingly, the LF82 strain of AIEC harbors four different RNRs (two class Ia, one class Ib and one class III). Although the E. coli RNR enzymes have been extensively characterized both biochemically and enzymatically, little is known about their roles during bacterial infection. We found that RNR expression was modified in AIEC LF82 bacteria during cell infection, suggesting that RNRs play an important role in AIEC virulence. Knockout of the nrdR and nrdD genes, which encodes a transcriptional regulator of RNRs and class III anaerobic RNR respectively, decreased AIEC LF82's ability to colonize the gut mucosa of transgenic mice that express human CEACAM6 (carcinoembryonic antigen-related cell-adhesion molecule 6). Microarray experiments demonstrated that NrdR plays an indirect role in AIEC virulence by interfering with bacterial motility and chemotaxis. Thus, the development of drugs targeting RNR classes, in particular NrdR and NrdD, could be a promising new strategy to control gut colonization by AIEC bacteria in CD patients.

Barreiros dos Santos, M., Azevedo, S., Agusil, J. P., Prieto-Simón, B., Sporer, C., Torrents, E., Juárez, A., Teixeira, V., Samitier, J., (2015). Label-free ITO-based immunosensor for the detection of very low concentrations of pathogenic bacteria Bioelectrochemistry , 101, 146-152

Abstract Here we describe the fabrication of a highly sensitive and label-free ITO-based impedimetric immunosensor for the detection of pathogenic bacteria Escherichia coli O157:H7. Anti-E. coli antibodies were immobilized onto ITO electrodes using a simple, robust and direct methodology. First, the covalent attachment of epoxysilane on the ITO surface was demonstrated by Atomic Force Microscopy and cyclic voltammetry. The immobilization of antibody on the epoxysilane layer was quantified by Optical Waveguide Lightmode Spectroscopy, obtaining a mass variation of 12 ng cm− 2 (0.08 pmol cm− 2). Microcontact printing and fluorescence microscopy were used to demonstrate the specific binding of E. coli O157:H7 to the antibody-patterned surface. We achieved a ratio of 1:500 Salmonella typhimurium/E. coli O157:H7, thus confirming the selectivity of the antibodies and efficiency of the functionalization procedure. Finally, the detection capacity of the ITO-based immunosensor was evaluated by Electrochemical Impedance Spectroscopy. A very low limit of detection was obtained (1 CFU mL− 1) over a large linear working range (10–106 CFU mL− 1). The specificity of the impedimetric immunosensor was also examined. Less than 20% of non-specific bacteria (S. typhimurium and E. coli K12) was observed. Our results reveal the applicability of ITO for the development of highly sensitive and selective impedimetric immunosensors.

Keywords: E. coli O157:H7, Electrochemical Impedance Spectroscopy, Immunosensor, Indium tin oxide, Label-free detection

Crespo, A., Pedraz, L., Torrents, E., (2015). Function of the Pseudomonas aeruginosa NrdR transcription factor: Global transcriptomic analysis and its role on ribonucleotide reductase gene expression PLoS ONE 10, (4), e0123571

Ribonucleotide reductases (RNRs) are a family of sophisticated enzymes responsible for the synthesis of the deoxyribonucleotides (dNTPs), the building blocks for DNA synthesis and repair. Although any living cell must contain one RNR activity to continue living, bacteria have the capacity to encode different RNR classes in the same genome, allowing them to adapt to different environments and growing conditions. Pseudomonas aeruginosa is well known for its adaptability and surprisingly encodes all three known RNR classes (Ia, II and III). There must be a complex transcriptional regulation network behind this RNR activity, dictating which RNR class will be expressed according to specific growing conditions. In this work, we aim to uncover the role of the transcriptional regulator NrdR in P. aeruginosa. We demonstrate that NrdR regulates all three RNR classes, being involved in differential control depending on whether the growth conditions are aerobic or anaerobic. Moreover, we also identify for the first time that NrdR is not only involved in controlling RNR expression but also regulates topoisomerase I (topA) transcription. Finally, to obtain the entire picture of NrdR regulon, we performed a global transcriptomic analysis comparing the transcription profile of wild-type and nrdR mutant strains. The results provide many new data about the regulatory network that controls P. aeruginosa RNR transcription, bringing us a step closer to the understanding of this complex system.

Julián, E., Baelo, A., Gavaldà, J., Torrents, E., (2015). Methyl-hydroxylamine as an efficacious antibacterial agent that targets the ribonucleotide reductase enzyme PLoS ONE 10, (3), e0122049

The emergence of multidrug-resistant bacteria has encouraged vigorous efforts to develop antimicrobial agents with new mechanisms of action. Ribonucleotide reductase (RNR) is a key enzyme in DNA replication that acts by converting ribonucleotides into the corresponding deoxyribonucleotides, which are the building blocks of DNA replication and repair. RNR has been extensively studied as an ideal target for DNA inhibition, and several drugs that are already available on the market are used for anticancer and antiviral activity. However, the high toxicity of these current drugs to eukaryotic cells does not permit their use as antibacterial agents. Here, we present a radical scavenger compound that inhibited bacterial RNR, and the compound's activity as an antibacterial agent together with its toxicity in eukaryotic cells were evaluated. First, the efficacy of N-methyl-hydroxylamine (M-HA) in inhibiting the growth of different Gram-positive and Gram-negative bacteria was demonstrated, and no effect on eukaryotic cells was observed. M-HA showed remarkable efficacy against Mycobacterium bovis BCG and Pseudomonas aeruginosa. Thus, given the M-HA activity against these two bacteria, our results showed that M-HA has intracellular antimycobacterial activity against BCG-infected macrophages, and it is efficacious in partially disassembling and inhibiting the further formation of P. aeruginosa biofilms. Furthermore, M-HA and ciprofloxacin showed a synergistic effect that caused a massive reduction in a P. aeruginosa biofilm. Overall, our results suggest the vast potential of M-HA as an antibacterial agent, which acts by specifically targeting a bacterial RNR enzyme.

Barniol-Xicota, M., Escandell, A., Valverde, E., Julián, E., Torrents, E., Vázquez, S., (2015). Antibacterial activity of novel benzopolycyclic amines Bioorganic and Medicinal Chemistry , 23, (2), 290-296

Staphylococcus aureus, especially strains resistant to multiple antibiotics, is a major pathogen for humans and animals. In this paper we have synthesized and evaluated the antibacterial activity of a new series of benzopolycyclic amines. Some of them exhibited μM MIC values against Staphylococcus aureus and other bacteria, including methicillin-resistant S. aureus MRSA. Compound 8 that displayed a good selectivity index, showed to be active in eliminating bacterial cells forming a preexisting biofilm.

Keywords: Antibacterials, Minimal biofilm inhibitory concentration, Polycyclic compounds, Staphylococcus aureus

Jaramillo, Maria del Carmen, Huttener, Mario, Alvarez, Juan Manuel, Homs-Corbera, Antoni, Samitier, Josep, Torrents, Eduard, Juárez, Antonio, (2015). Dielectrophoresis chips improve PCR detection of the food-spoiling yeast Zygosaccharomyces rouxii in apple juice Electrophoresis , 36, (13), 1471-1478

DEP manipulation of cells present in real samples is challenging. We show in this work that an interdigitated DEP chip can be used to trap and wash a population of the food-spoiling yeast Zygosaccharomyces rouxii that contaminates a sample of apple juice. By previously calibrating the chip, the yeast population loaded is efficiently trapped, washed and recovered in a small-volume fraction which, in turn, can be used for efficient PCR detection of this yeast. DEP washing of yeast cells gets rid of PCR inhibitors present in apple juice and facilitates PCR analysis. This and previous works on the use of DEP chips to improve PCR analysis show that a potential use of DEP is to be used as a treatment of real samples prior to PCR.

Keywords: Dielectrophoresis, PCR, Saccharomyces, Yeast

Basas, J., Rojo, E., Gomis, X., Sierra, J.M., Torrents, E., Almirante, B., Gavaldà, J., (2015). Actividad de anidulafungia vs. anfotericina b liposomal frente a C. Parapsilopsis creciendo en biopelículas en distintos materiales Enfermedades Infecciosas y Microbiología Clínica XIX Congreso de la Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica (SEIMC) , Elsevier (Sevilla, Spain) 33, 37

La infección del catéter venoso central está estrechamente relacionada con la capacidad de los microorganismos para producir biopelículas. Existen metodologías distintas para el estudio in vitro de la sensibilidad antibiótica de microorganismos creciendo en biopelículas; con placas de microtitulación (poliestireno) y con diferentes materiales (discos silicona, placas titanio...). Se ha descrito que las especies de Candida crecen con morfologías diferentes dependiendo del sustrato donde se implantan. Por tanto, la elección del material donde crecen las biopelículas podría tener su importancia en los estudios de sensibilidad in vitro. Previamente, en un modelo experimental de infección de catéter por C. parapsilopsis observamos que el sellado con anidulafungina (And) era más eficaz que con anfotericina B liposomal (LAmB). Teniendo en cuenta estas consideraciones el mejor sustrato para valorar la eficacia in vitro sería en discos de silicona (utilizado para la fabricación de catéteres) y no en placas de microtitulación (técnica estándar).

Torrents, E., (2015). Tratamientos antimicrobianos dirigidos. ¿Es posible la nanomedicina en las enfermedades infecciosas? Enfermedades Infecciosas y Microbiología Clínica XIX Congreso de la Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica (SEIMC) , Elsevier (Sevilla, Spain) 33, 6

A lo largo del siglo XX, los avances en el desarrollo de los antibióticos han jugado un papel de gran importancia en la lucha contra las enfermedades infecciosas. Sin embargo, el uso inadecuado de éstos está conduciendo a la aparición de resistencias a múltiples fármacos (multidrug resistance, MDR) en diversos patógenos. El gran costo y complejidad asociados al descubrimiento de nuevos fármacos agrava la situación, propiciando que un número muy reducido de nuevos antibióticos se haya descubierto en los últimos 40 años. Esta situación provocará, si no se toman acciones para evitarlo, un gran problema de salud pública global durante el siglo XXI. En la última década se ha producido, en cambio, un gran avance en el campo de la nanotecnología, permitiendo el diseño de nanopartículas con propiedades fisicoquímicas deseables para su uso en microbiología. La escala de estas partículas ofrece un gran incremento en su relación superficie/volumen respecto a otras formas de liberación de fármacos, lo que ha permitido reconsiderar el uso de antiguas sustancias antimicrobianas, como la plata, el cobre o el zinc. Las nanopartículas se están así proyectando como una nueva línea de defensa contra los patógenos bacterianos, en especial los multirresistentes. Se comentarán los avances recientes en el diseño de nanopartículas, demostrando el potencial de éstas en la lucha contra las infecciones bacterianas. Igualmente, se comentarán nuevas estrategias, como la combinación diferentes fármacos y/o antibióticos encapsulados en nanopartículas (nanoantibióticos). Por último, explicaremos nuestra propia experiencia en el uso de nanopartículas de PLGA, o ácido poli(láctico-co-glicólico) modificadas, para la lucha contra la bacteria Pseudomonas aeruginosa creciendo en forma de biofilm.

Cendra, M. M., Torrents, E., (2014). Enzims essencials per a la vida Treballs de la Societat Catalana de Biologia , 65, 64-67

Les ribonucleòtid-reductases (RNR) són enzims essencials per a tota cèllula, perquè fan la transformació dels ribonucleòtids a desoxiribonucleòtids, els quals són necessaris per a la síntesi de l’àcid desoxiribonucleic (DNA). És evident que les RNR són enzims ancestrals i clau en l’evolució del material genètic que hi ha actualment, i són essencials per a l’evolució de tots els organismes que hi ha sobre la Terra. A causa de l’essencialitat de la reacció que fan aquests enzims, es poden considerar una diana ideal per al disseny de compostos que inhibeixen la replicació cel·lular, ja sigui en cèl·lules eucariòtiques (incloent-hi cèl·lules cancerígenes), com agents bacterians infecciosos.

Oliva, A. M., Homs-Corbera, A., Torrents, E., Juarez, A., Samitier, J., (2014). Synergystic effect of temperature and electric field intensity in Escherichia coli inactivation Micro and Nanosystems , 6, (2), 79-86

Electric Fields are increasingly used to manipulate bacteria. However, there is no systematic and definitive study on how the different electric parameters change bacteria viability. Here we present a study on the effects of electric field intensity and temperature to bacterial cultures. Escherichia coli colonies have been exposed to different electric field intensities at 1MHz during 5 minutes by means of a microfluidic device specially designed for the experiment. From the analysis of the results it is possible to see that Escherichia coli survival rate diminishes when applying field intensities as low as 220V during 5 minutes. Death rates also increase when stronger fields are applied. However, viability of survived bacteria is maintained. Additionally, temperature shows a synergistic effect with voltage. When temperature was increased, results showed a stronger sensitivity of cells to the electric field. Moreover, the expression patterns of Outer Membrane Protein A and Ribosomal Proteins differ in control and treated samples, suggesting changes in bacterial metabolism and structure.

Keywords: E. coli, Electric field, Temperature, Viability

Torrents, Eduard, (2014). Ribonucleotide reductases: Essential Enzymes for bacterial life Frontiers in Cellular and Infection Microbiology , 4, 1-9

Ribonucleotide reductase (RNR) is a key enzyme that mediates the synthesis of deoxyribonucleotides, the DNA precursors, for DNA synthesis in every living cell. This enzyme converts ribonucleotides to deoxyribonucleotides, the building blocks for DNA replication, and repair. Clearly, RNR enzymes have contributed to the appearance of genetic material that exists today, being essential for the evolution of all organisms on Earth. The strict control of RNR activity and dNTP pool sizes is important, as pool imbalances increase mutation rates, replication anomalies, and genome instability. Thus, RNR activity should be finely regulated allosterically and at the transcriptional level. In this review we examine the distribution, the evolution, and the genetic regulation of bacterial RNRs. Moreover, this enzyme can be considered an ideal target for anti-proliferative compounds designed to inhibit cell replication in eukaryotic cells (cancer cells), parasites, viruses, and bacteria.

Keywords: Anaerobiosis, Transcription Factors, Evolution, Gene regulation, Ribonucleotide reductase, DNA Synthesis, NrdR,nrd

Oliva, A. M., Homs, A., Torrents, E., Juarez, A., Samitier, J., (2014). Effect of electric field and temperature in E.Coli viability IFMBE Proceedings XIII Mediterranean Conference on Medical and Biological Engineering and Computing 2013 (ed. Roa Romero, Laura M.), Springer (Seville, Spain) 41, 1833-1836

Electromagnetic Fields are increasingly used to manipulate bacteria. However, there is no systematic and definitive study on how the different electric parameters change bacteria viability. Here we present preliminary data on the effect of electric field intensity and temperature applica- tion. E. Coli colonies have been exposed to different voltages at 1MHz during 5 minutes by means of a custom-made micro- fluidic device. Results show that E.Coli survival rate is already reduced by applying field intensities as low as 220V/cm during 5 minutes. The use of stronger fields resulted in death rates increase also. Viability of survived bacteria was maintained. On the other hand, temperature has shown a synergistic effect with voltage. When temperature is increased results seem to indicate stronger sensitivity of cells to the electric field. It is necessary to continue studying the contribution of other para- meters as intensity, time, frequency or concentration, to study further synergies.

Keywords: E. Coli, Electromagnetic Field, Temperature, Viability

Jaramillo, M. C., Martínez-Duarte, R., Hüttener, M., Renaud, P., Torrents, E., Juárez, A., (2013). Increasing PCR sensitivity by removal of polymerase inhibitors in environmental samples by using dielectrophoresis Biosensors and Bioelectronics 43, (1), 297-303

Dielectrophoresis (DEP) is a powerful tool to manipulate cells and molecules in microfluidic chips. However, few practical applications using DEP exist. An immediate practical application of a carbon-electrode DEP system, in removing PCR inhibitors from a sample, is reported in this work. We use a high throughput carbon-electrode DEP system to trap yeast cells from a natural sample (fermented grape must) and then in situ remove contaminants that interfere with PCR analysis. Retrieval of this enriched and purified yeast population from the DEP system then allows for a significant increase of sensitivity during PCR analysis. Furthermore, the fact that DEP can discriminate between viable and non-viable cells minimizes the number of false positives commonly obtained when using PCR alone. Experimental results provide clear evidence that the carbon-electrode DEP-based sample preparation step can readily and effectively clean environmental samples from natural contaminants and improve PCR sensitivity.

Otero, J., Baños, R., González, L., Torrents, E., Juárez, A., Puig-Vidal, M., (2013). Quartz tuning fork studies on the surface properties of Pseudomonas aeruginosa during early stages of biofilm formation Colloids and Surfaces B: Biointerfaces 102, 117-123

Scanning probe microscopy techniques are powerful tools for studying the nanoscale surface properties of biofilms, such as their morphology and mechanical behavior. Typically, these studies are conducted using atomic force microscopy probes, which are force nanosensors based on microfabricated cantilevers. In recent years, quartz tuning fork (QTF) probes have been used in morphological studies due to their better performance in certain experiments with respect to standard AFM probes. In the present work QTF probes were used to measure not only the morphology but also the nanomechanical properties of Pseudomonas aeruginosa during early stages of biofilm formation. Changes in bacterium size and the membrane spring constant were determined in biofilms grown for 20, 24 and 28. h on gold with and without glucose in the culture media. The results obtained using the standard AFM and QTF probes were compared. Both probes showed that the bacteria forming the biofilm increased in size over time, but that there was no dependence on the presence of glucose in the culture media. On the other hand, the spring constant increased over time and there was a clear difference between biofilms grown with and without glucose. This is the first time that QTF probes have been used to measure the nanomechanical properties of microbial cell surfaces and the results obtained highlight their potential for studying biological samples beyond topographic measurements.

Cendra, M. M., Juárez, A., Madrid, C., Torrents, E., (2013). H-NS is a novel transcriptional modulator of the ribonucleotide reductase genes in escherichia coli Journal of Bacteriology , 195, (18), 4255-4263

Ribonucleotide reductases (RNRs) are essential enzymes for DNA synthesis because they are responsible for the production of the four deoxyribonucleotides (dNTPs) from their corresponding ribonucleotides. Escherichia coli contains two classes of aerobic RNRs, encoded by the nrdAB (class Ia) and nrdHIEF (class Ib) operons, and a third RNR class, which is functional under anaerobic conditions and is encoded by the nrdDG (class III) operon. Because cellular imbalances in the amounts of the four dNTPs cause an increase in the rate of mutagenesis, the activity and the expression of RNRs must be tightly regulated during bacterial chromosome replication. The transcriptional regulation of these genes requires several transcription factors (including DnaA, IciA, FIS [factor for inversion stimulation], Fnr, Fur, and NrdR), depending on the RNR class; however, the factors that dictate the expression of some RNR genes in response to different environmental conditions are not known. We show that H-NS modulates the expression of the nrdAB and nrdDG operons. H-NS represses expression both in aerobically and in anaerobically growing cells. Under aerobic conditions, repression occurs at the exponential phase of growth as well as at the transition from the exponential to the stationary phase, a period when no dNTPs are needed. Under anoxic conditions, repression occurs mainly in exponentially growing cells. Electrophoretic mobility assays performed with two DNA fragments from the regulatory region of the nrdAB operon demonstrated the direct interaction of H-NS with these sequences.

Cendra, M. d M., Juárez, A., Torrents, E., (2012). Biofilm modifies expression of ribonucleotide reductase genes in Escherichia coli PLoS ONE 7, (9), e46350

Ribonucleotide reductase (RNR) is an essential enzyme for all living organisms since is the responsible for the last step in the synthesis of the four deoxyribonucleotides (dNTPs) necessary for DNA replication and repair. In this work, we have investigated the expression of the three-RNR classes (Ia, Ib and III) during Escherichia coli biofilm formation. We show the temporal and spatial importance of class Ib and III RNRs during this process in two different E. coli wild-type strains, the commensal MG1655 and the enteropathogenic and virulent E2348/69, the prototype for the enteropathogenic E. coli (EPEC). We have established that class Ib RNR, so far considered cryptic, play and important role during biofilm formation. The implication of this RNR class under the specific growth conditions of biofilm formation is discussed.

Crona, Mikael, Torrents, Eduard, Rohr, Asmund K., Hofer, Anders, Furrer, Ernst, Tomter, Ane B., Andersson, K. Kristoffer, Sahlin, Margareta, Sjoberg, Britt-Marie, (2011). NrdH-redoxin protein mediates high enzyme activity in manganese-reconstituted ribonucleotide reductase from bacillus anthracis Journal of Biological Chemistry , 286, (38), 33053-33060

Bacillus anthracis is a severe mammalian pathogen encoding a class Ib ribonucleotide reductase (RNR). RNR is a universal enzyme that provides the four essential deoxyribonucleotides needed for DNA replication and repair. Almost all Bacillus spp. encode both class Ib and class III RNR operons, but the B. anthracis class III operon was reported to encode a pseudogene, and conceivably class Ib RNR is necessary for spore germination and proliferation of B. anthracis upon infection. The class Ib RNR operon in B. anthracis encodes genes for the catalytic NrdE protein, the tyrosyl radical metalloprotein NrdF, and the flavodoxin protein NrdI. The tyrosyl radical in NrdF is stabilized by an adjacent Mn(2)(III) site (Mn-NrdF) formed by the action of the NrdI protein or by a Fe(2)(III) site (Fe-NrdF) formed spontaneously from Fe(2+) and O(2). In this study, we show that the properties of B. anthracis Mn-NrdF and Fe-NrdF are in general similar for interaction with NrdE and NrdI. Intriguingly, the enzyme activity of Mn-NrdF was approximately an order of magnitude higher than that of Fe-NrdF in the presence of the class Ib-specific physiological reductant NrdH, strongly suggesting that the Mn-NrdF form is important in the life cycle of B. anthracis. Whether the Fe-NrdF form only exists in vitro or whether the NrdF protein in B. anthracis is a true cambialistic enzyme that can work with either manganese or iron remains to be established.

Keywords: Escherichia-coli, Corynebacterium-ammoniagenes, Crystal-structure, Cofactor, Cubunit, Growth, Genes

Sjoberg, B. M., Torrents, E., (2011). Shift in ribonucleotide reductase gene expression in pseudomonas aeruginosa during infection Infection and Immunity , 79, (7), 2663-2669

The roles of different ribonucleotide reductases (RNRs) in bacterial pathogenesis have not been studied systematically. In this work we analyzed the importance of the different Pseudomonas aeruginosa RNRs in pathogenesis using the Drosophila melanogaster host-pathogen interaction model. P. aeruginosa codes for three different RNRs with different environmental requirements. Class II and III RNR chromosomal mutants exhibited reduced virulence in this model. Translational reporter fusions of RNR gene nrdA, nrdJ, or nrdD to the green fluorescent protein were constructed to measure the expression of each class during the infection process. Analysis of the P. aeruginosa infection by flow cytometry revealed increased expression of nrdJ and nrdD and decreased nrdA expression during the infection process. Expression of each RNR class fits with the pathogenicities of the chromosomal deletion mutants. An extended understanding of the pathogenicity and physiology of P. aeruginosa will be important for the development of novel drugs against infections in cystic fibrosis patients.

Keywords: Broad-host-range, Anaerobic growth, Drosophila-melanogaster, Bacterial biofilms, Escherichia-coli, Cystic-fibrosis, Model host, Virulence, Promoter, Vectors

Adrados, B., Julian, E., Codony, F., Torrents, E., Luquin, M., Morato, J., (2011). Prevalence and concentration of non-tuberculous Mycobacteria in cooling towers by means of quantitative PCR: A prospective study Current Microbiology , 62, (1), 313-319

There is an increasing level of interest in non-tuberculous mycobacteria (NTM) due to the increasing reported rates of diseases caused by them. Although it is well known that NTM are widely distributed in the environment it is necessary to identify its reservoirs to prevent possible infections. In this study, we aimed to investigate the occurrence and levels of NTM in cooling towers to provide evidences for considering these settings as possible sources of respiratory infections. In the current study, we detected and quantified the presence of NTM by means of a rapid method in water samples taken from 53 cooling towers of an urban area (Barcelona, Spain). A genus-specific quantitative PCR (Q-PCR) assay with a quantification limit (QL) of 500 cells l(-1) was used. 56% (30) of samples were positive with a concentration range from 4.6 x 10(3) to 1.79 x 10(6) cells l(-1). In some cases (9/30), samples were positive but with levels below the QL. The colonization rate confirmed that cooling towers could be considered as a potential reservoir for NTM. This study also evaluated Q-PCR as a useful method to detect and quantify NTM in samples coming from environmental sources.

Keywords: Real-time PCR, Disease, Identification, Tuberculosis, Pathogens, Waters

Lundin, Daniel, Gribaldo, Simonetta, Torrents, Eduard, Sjoberg, Britt-Marie, Poole, Anthony, (2010). Ribonucleotide reduction - horizontal transfer of a required function spans all three domains BMC Evolutionary Biology , 10, (1), 383

BACKGROUND:Ribonucleotide reduction is the only de novo pathway for synthesis of deoxyribonucleotides, the building blocks of DNA. The reaction is catalysed by ribonucleotide reductases (RNRs), an ancient enzyme family comprised of three classes. Each class has distinct operational constraints, and are broadly distributed across organisms from all three domains, though few class I RNRs have been identified in archaeal genomes, and classes II and III likewise appear rare across eukaryotes. In this study, we examine whether this distribution is best explained by presence of all three classes in the Last Universal Common Ancestor (LUCA), or by horizontal gene transfer (HGT) of RNR genes. We also examine to what extent environmental factors may have impacted the distribution of RNR classes.RESULTS:Our phylogenies show that the Last Eukaryotic Common Ancestor (LECA) possessed a class I RNR, but that the eukaryotic class I enzymes are not directly descended from class I RNRs in Archaea. Instead, our results indicate that archaeal class I RNR genes have been independently transferred from bacteria on two occasions. While LECA possessed a class I RNR, our trees indicate that this is ultimately bacterial in origin. We also find convincing evidence that eukaryotic class I RNR has been transferred to the Bacteroidetes, providing a stunning example of HGT from eukaryotes back to Bacteria. Based on our phylogenies and available genetic and genomic evidence, class II and III RNRs in eukaryotes also appear to have been transferred from Bacteria, with subsequent within-domain transfer between distantly-related eukaryotes. Under the three-domains hypothesis the RNR present in the last common ancestor of Archaea and eukaryotes appears, through a process of elimination, to have been a dimeric class II RNR, though limited sampling of eukaryotes precludes a firm conclusion as the data may be equally well accounted for by HGT.CONCLUSIONS:Horizontal gene transfer has clearly played an important role in the evolution of the RNR repertoire of organisms from all three domains of life. Our results clearly show that class I RNRs have spread to Archaea and eukaryotes via transfers from the bacterial domain, indicating that class I likely evolved in the Bacteria. However, against the backdrop of ongoing transfers, it is harder to establish whether class II or III RNRs were present in the LUCA, despite the fact that ribonucleotide reduction is an essential cellular reaction and was pivotal to the transition from RNA to DNA genomes. Instead, a general pattern of ongoing horizontal transmission emerges wherein environmental and enzyme operational constraints, especially the presence or absence of oxygen, are likely to be major determinants of the RNR repertoire of genomes.

Keywords: -----

Torrents, E., Sjoberg, B. M., (2010). Antibacterial activity of radical scavengers against class Ib ribonucleotide reductase from Bacillus anthracis Biological Chemistry , 391, (2-3), 229-234

Bacillus anthracis is a severe mammalian pathogen. The deoxyribonucleotides necessary for DNA replication and repair are provided via the ribonucleotide reductase (RNR) enzyme. RNR is also important for spore germination and cell proliferation upon infection. We show that the expression of B. anthracis class Ib RNR responds to the environment that the pathogen encounters upon infection. We also show that several anti-proliferative agents (radical scavengers) specifically inhibit the B. anthracis RNR. Owing to the importance of RNR in the pathogenic infection process, our results highlight a promising potential to inhibit the growth of B. anthracis early during infection.

Keywords: Anthrax, Antibacterial drug, Antibacterial target, Enzyme inhibition

Jaramillo, M. D., Torrents, E., Martinez-Duarte, R., Madou, M. J., Juarez, A., (2010). On-line separation of bacterial cells by carbon-electrode dielectrophoresis Electrophoresis , 31, (17), 2921-2928

Dielectrophoresis (DEP) represents a powerful approach to manipulate and study living cells. Hitherto, several approaches have used 2-D DEP chips. With the aim to increase sample volume, in this study we used a 3-D carbon-electrode DEP chip to trap and release bacterial cells. A continuous flow was used to plug an Escherichia coli cell suspension first, to retain cells by positive DEP, and thereafter to recover them by washing with peptone water washing solution. This approach allows one not only to analyze DEP behavior of living cells within the chip, but also to further recover fractions containing DEP-trapped cells. Bacterial concentration and flow rate appeared as critical parameters influencing the separation capacity of the chip. Evidence is presented demonstrating that the setup developed in this study can be used to separate different types of bacterial cells.

Keywords: Bacteria, Carbon electrode, Dielectrophoresis, E. coli, Separation

Johansson, R., Torrents, E., Lundin, D., Sprenger, J., Sahlin, M., Sjöberg, B. M., Logan, D. T., (2010). High-resolution crystal structures of the flavoprotein NrdI in oxidized and reduced states – an unusual flavodoxin FEBS Journal , 277, (20), 4265-4277

The small flavoprotein NrdI is an essential component of the class Ib ribonucleotide reductase system in many bacteria. NrdI interacts with the class Ib radical generating protein NrdF. It is suggested to be involved in the rescue of inactivated diferric centres or generation of active dimanganese centres in NrdF. Although NrdI bears a superficial resemblance to flavodoxin, its redox properties have been demonstrated to be strikingly different. In particular, NrdI is capable of two-electron reduction, whereas flavodoxins are exclusively one-electron reductants. This has been suggested to depend on a lesser destabilization of the negatively-charged hydroquinone state than in flavodoxins. We have determined the crystal structures of NrdI from Bacillus anthracis, the causative agent of anthrax, in the oxidized and semiquinone forms, at resolutions of 0.96 and 1.4 Å, respectively. These structures, coupled with analysis of all curated NrdI sequences, suggest that NrdI defines a new structural family within the flavodoxin superfamily. The conformational behaviour of NrdI in response to FMN reduction is very similar to that of flavodoxins, involving a peptide flip in a loop near the N5 atom of the flavin ring. However, NrdI is much less negatively charged than flavodoxins, which is expected to affect its redox properties significantly. Indeed, sequence analysis shows a remarkable spread in the predicted isoelectric points of NrdIs, from approximately pH 4–10. The implications of these observations for class Ib ribonucleotide reductase function are discussed.

Keywords: Crystal structure, Flavin mononucleotide, Flavodoxin, NrdI, Ribonucleotide reductase

Lundin, Daniel, Torrents, Eduard, Poole, Anthony, Sjoberg, Britt-Marie, (2009). RNRdb, a curated database of the universal enzyme family ribonucleotide reductase, reveals a high level of misannotation in sequences deposited to Genbank BMC Genomics 10, (1), 589

BACKGROUND:Ribonucleotide reductases (RNRs) catalyse the only known de novo pathway for deoxyribonucleotide synthesis, and are therefore essential to DNA-based life. While ribonucleotide reduction has a single evolutionary origin, significant differences between RNRs nevertheless exist, notably in cofactor requirements, subunit composition and allosteric regulation. These differences result in distinct operational constraints (anaerobicity, iron/oxygen dependence and cobalamin dependence), and form the basis for the classification of RNRs into three classes.DESCRIPTION:In RNRdb (Ribonucleotide Reductase database), we have collated and curated all known RNR protein sequences with the aim of providing a resource for exploration of RNR diversity and distribution. By comparing expert manual annotations with annotations stored in Genbank, we find that significant inaccuracies exist in larger databases. To our surprise, only 23% of protein sequences included in RNRdb are correctly annotated across the key attributes of class, role and function, with 17% being incorrectly annotated across all three categories. This illustrates the utility of specialist databases for applications where a high degree of annotation accuracy may be important. The database houses information on annotation, distribution and diversity of RNRs, and links to solved RNR structures, and can be searched through a BLAST interface. RNRdb is accessible through a public web interface at is a specialist database that provides a reliable annotation and classification resource for RNR proteins, as well as a tool to explore distribution patterns of RNR classes. The recent expansion in available genome sequence data have provided us with a picture of RNR distribution that is more complex than believed only a few years ago; our database indicates that RNRs of all three classes are found across all three cellular domains. Moreover, we find a number of organisms that encode all three classes.

Keywords: Enzymology (Biochemistry and Molecular Biophysics), Computer Applications (Computational Biology)

Torrents, E., Sahlin, M., Sjöberg, B., (2009). The ribonucleotide reductase family: genetics and genomics Nova Biomedical (ed. Andersson, K.K.), Nova Science Publishers (New York, USA) , 99


Keywords: -----

Roca, Ignasi, Torrents, Eduard, Sahlin, Margareta, Gibert, Isidre, Sjoberg, Britt-Marie, (2008). NrdI essentiality for class Ib ribonucleotide reduction in streptococcus pyogenes Journal of Bacteriology , 190, (14), 4849-4858

The Streptococcus pyogenes genome harbors two clusters of class Ib ribonucleotide reductase genes, nrdHEF and nrdF*I*E*, and a second stand-alone nrdI gene, designated nrdI2. We show that both clusters are expressed simultaneously as two independent operons. The NrdEF enzyme is functionally active in vitro, while the NrdE*F* enzyme is not. The NrdF* protein lacks three of the six highly conserved iron-liganding side chains and cannot form a dinuclear iron site or a tyrosyl radical. In vivo, on the other hand, both operons are functional in heterologous complementation in Escherichia coli. The nrdF*I*E* operon requires the presence of the nrdI* gene, and the nrdHEF operon gained activity upon cotranscription of the heterologous nrdI gene from Streptococcus pneumoniae, while neither nrdI* nor nrdI2 from S. pyogenes rendered it active. Our results highlight the essential role of the flavodoxin NrdI protein in vivo, and we suggest that it is needed to reduce met-NrdF, thereby enabling the spontaneous reformation of the tyrosyl radical. The NrdI* flavodoxin may play a more direct role in ribonucleotide reduction by the NrdF*I*E* system. We discuss the possibility that the nrdF*I*E* operon has been horizontally transferred to S. pyogenes from Mycoplasma spp.

Keywords: Group-a streptococcus, Bacillus-subtilis genes, Escherichia-coli, Corynebacterium-ammoniagenes, Mycobacterium-tuberculosis, Expression analysis, Genome sequence, Small-subunit, Salmonella-typhimurium, Iron center