Staff member publications
García-Díaz, María, Cendra, Maria del Mar, Alonso-Roman, Raquel, Urdániz, María, Torrents, Eduard, Martínez, Elena, (2022). Mimicking the Intestinal Host–Pathogen Interactions in a 3D In Vitro Model: The Role of the Mucus Layer Pharmaceutics 14, 1552
The intestinal mucus lines the luminal surface of the intestinal epithelium. This mucus is a dynamic semipermeable barrier and one of the first-line defense mechanisms against the outside environment, protecting the body against chemical, mechanical, or biological external insults. At the same time, the intestinal mucus accommodates the resident microbiota, providing nutrients and attachment sites, and therefore playing an essential role in the host–pathogen interactions and gut homeostasis. Underneath this mucus layer, the intestinal epithelium is organized into finger-like protrusions called villi and invaginations called crypts. This characteristic 3D architecture is known to influence the epithelial cell differentiation and function. However, when modelling in vitro the intestinal host–pathogen interactions, these two essential features, the intestinal mucus and the 3D topography are often not represented, thus limiting the relevance of the models. Here we present an in vitro model that mimics the small intestinal mucosa and its interactions with intestinal pathogens in a relevant manner, containing the secreted mucus layer and the epithelial barrier in a 3D villus-like hydrogel scaffold. This 3D architecture significantly enhanced the secretion of mucus. In infection with the pathogenic adherent invasive E. coli strain LF82, characteristic of Crohn’s disease, we observed that this secreted mucus promoted the adhesion of the pathogen and at the same time had a protective effect upon its invasion. This pathogenic strain was able to survive inside the epithelial cells and trigger an inflammatory response that was milder when a thick mucus layer was present. Thus, we demonstrated that our model faithfully mimics the key features of the intestinal mucosa necessary to study the interactions with intestinal pathogens.
JTD Keywords: barrier function, bile-salts, cells, drug-delivery, host-pathogen interaction, hydrogels, ileal mucosa, infection, intestinal models, intestinal mucus, microbiome, patient, responses, 3d in vitro models, Invasive escherichia-coli
Vukomanovic M, Cendra MdM, Baelo A, Torrents E, (2021). Nano-engineering stable contact-based antimicrobials: Chemistry at the interface between nano-gold and bacteria Colloids And Surfaces B-Biointerfaces 208, 112083
Contact-based antimicrobials, as antibiotic-free technologies that use non-specific interactions with bacterial cells to exert antimicrobial activity, are a prospective solution in fighting the global issue of bacterial resistance. A very simplified approach to their design considers the direct bonding of cationic guanidine-containing amino acids to the surface of nano-gold carriers. The structure enables antimicrobial activity due to a high density of cationic surface charges. This opens a set of novel questions that are important for their effective engineering, particularly regarding (i) chemistry and events that take place at the interface between NPs and cells, (ii) the direct influence of a charge (and its change) on interactions with bacterial and mammalian cells, and (iii) the stability of structures (and their antimicrobial activity) in the presence of enzymes, which are addressed in this paper. Because of the ability of amino acid-functionalized nano-gold to retain structural and functional activity, even after exposure to a range of physicochemical stimuli, they provide an excellent nanotechnological platform for designing highly effective contact-based antimicrobials and their applications.
JTD Keywords: agents, antibiotic-free technology, arginine, charged amino acids, contact-based antimicrobials, discovery, enzyme-resistant antimicrobials, functionalized gold, peptides, polymers, resistant, Antibiotic-free technology, Charged amino acids, Contact-based antimicrobials, Enzyme-resistant antimicrobials, Functionalized gold, Nanoparticles
Cendra MdM, Torrents E, (2021). Pseudomonas aeruginosa biofilms and their partners in crime Biotechnology Advances 49, 107734
Pseudomonas aeruginosa biofilms and the capacity of the bacterium to coexist and interact with a broad range of microorganisms have a substantial clinical impact. This review focuses on the main traits of P. aeruginosa biofilms, such as the structural composition and regulatory networks involved, placing particular emphasis on the clinical challenges they represent in terms of antimicrobial susceptibility and biofilm infection clearance. Furthermore, the ability of P. aeruginosa to grow together with other microorganisms is a significant pathogenic attribute with clinical relevance; hence, the main microbial interactions of Pseudomonas are especially highlighted and detailed throughout this review. This article also explores the infections caused by single and polymicrobial biofilms of P. aeruginosa and the current models used to recreate them under laboratory conditions. Finally, the antimicrobial and antibiofilm strategies developed against P. aeruginosa mono and multispecies biofilms are detailed at the end of this review.
JTD Keywords: aeruginosa models, antibiotic-resistance, antimicrobials, bacterial biofilms, biofilms, c-di-gmp, chronic infections, enterococcus-faecalis, extracellular dna, in-vitro, lectin pa-iil, p, p. aeruginosa models, polymicrobial, polymicrobial interactions, staphylococcus-aureus, Antimicrobials, Biofilms, Chronic infections, P. aeruginosa models, Polymicrobial, Pseudomonas aeruginosa, Urinary-tract-infection
Campo-Pérez V, Cendra MdM, Julián E, Torrents E, (2021). Easily applicable modifications to electroporation conditions improve the transformation efficiency rates for rough morphotypes of fast-growing mycobacteria New Biotechnology 63, 10-18
© 2021 Elsevier B.V. Electroporation is the most widely used and efficient method to transform mycobacteria. Through this technique, fast- and slow-growing mycobacteria with smooth and rough morphotypes have been successfully transformed. However, transformation efficiencies differ widely between species and strains. In this study, the smooth and rough morphotypes of Mycobacteroides abscessus and Mycolicibacterium brumae were used to improve current electroporation procedures for fast-growing rough mycobacteria. The focus was on minimizing three well-known and challenging limitations: the mycobacterial restriction-modification systems, which degrade foreign DNA; clump formation of electrocompetent cells before electroporation; and electrical discharges during pulse delivery, which were reduced by using salt-free DNA solution. Herein, different strategies are presented that successfully address these three limitations and clearly improve the electroporation efficiencies over the current procedures. The results demonstrated that combining the developed strategies during electroporation is highly recommended for the transformation of fast-growing rough mycobacteria.
JTD Keywords: clump, desalted dna, electroporation, mycobacteria, mycobacteroides abscessus, mycolicibacterium brumae, Clump, Desalted dna, Electroporation, Mycobacteria, Mycobacteroides abscessus, Mycolicibacterium brumae
Moya-Andérico L, Vukomanovic M, Cendra MdM, Segura-Feliu M, Gil V, del Río JA, Torrents E, (2021). Utility of Galleria mellonella larvae for evaluating nanoparticle toxicology Chemosphere 266, 129235
© 2020 Elsevier Ltd The use of nanoparticles in consumer products is currently on the rise, so it is important to have reliable methods to predict any associated toxicity effects. Traditional in vitro assays fail to mimic true physiological responses of living organisms against nanoparticles whereas murine in vivo models are costly and ethically controversial. For these reasons, this study aimed to evaluate the efficacy of Galleria mellonella as an alternative, non-rodent in vivo model for examining nanoparticle toxicity. Silver, selenium, and functionalized gold nanoparticles were synthesized, and their toxicity was assessed in G. mellonella larvae. The degree of acute toxicity effects caused by each type of NP was efficiently detected by an array of indicators within the larvae: LD50 calculation, hemocyte proliferation, NP distribution, behavioral changes, and histological alterations. G. mellonella larvae are proposed as a nanotoxicological model that can be used as a bridge between in vitro and in vivo murine assays in order to obtain better predictions of NP toxicity.
JTD Keywords: cellular uptake, cytotoxicity, galleria mellonella, gold nanoparticles, hemocytes, nanoparticles, nanotoxicity, non-rodent in vivo model, non-rodent in vivo model, oxidative stress, selenium-compounds, silica nanoparticles, silver nanoparticles, toxicity, toxicity screening, vitro, Galleria mellonella, Hemocytes, In-vivo model, Nanoparticles, Nanotoxicity, Non-rodent in vivo model, Toxicity screening
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.
JTD Keywords: Pseudomonas aeruginosa, Intracellular persistence, Lung, Epithelial cells, Clinical isolates, Host-pathogen interactions, Intracellular lifestyle, Chronic infections, Cystic fibrosis, Ribonucleotide reductase
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.
JTD Keywords: Applied microbiology, Biofilms
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.
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.
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.
Garcia, J., Madrid, C., Cendra, M., Juarez, A., Pons, M., (2009). N9L and L9N mutations toggle Hha binding and hemolysin regulation by Escherichia coli and Vibrio cholerae H-NS FEBS Letters , 583, (17), 2911-2916
Proteins of the Hha/YmoA family co-regulate with H-NS the expression of virulence factors in Enterobacteriaceae. Vibrio cholerae lacks Hha-like proteins and its H-NS (vcH-NS) is unable to bind Hha, in spite of the conservation of a key residue for Hha binding by Escherichia coli H-NS (ecH-NS). Exchange of the residues in position 9 between vcH-NS and ecH-NS strongly reduces Hha binding by ecH-NS and introduces it in vcH- NS. These mutations strongly affect the repression of the hemolysin operon in E. coli and the electrophoretic mobility of complexes formed with a DNA fragment containing its regulatory region.
JTD Keywords: Nucleoid associated protein, H-NS, Hha, Transcription repression, NMR, Electrophoretic mobility shift assays