Person: Toledo Arana, Alejandro
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Toledo Arana
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Alejandro
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Instituto de Agrobiotecnología (IdAB)
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0000-0001-8148-6281
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5497
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Publication Open Access Bacterial biofilm functionalization through Bap amyloid engineering(Springer Nature, 2022) Matilla Cuenca, Leticia; Taglialegna, Agustina; Gil Puig, Carmen; Toledo Arana, Alejandro; Lasa Uzcudun, Íñigo; Valle Turrillas, Jaione; Ciencias de la Salud; Osasun ZientziakBiofilm engineering has emerged as a controllable way to fabricate living structures with programmable functionalities. The amyloidogenic proteins comprising the biofilms can be engineered to create self-assembling extracellular functionalized surfaces. In this regard, facultative amyloids, which play a dual role in biofilm formation by acting as adhesins in their native conformation and as matrix scaffolds when they polymerize into amyloid-like fibrillar structures, are interesting candidates. Here, we report the use of the facultative amyloid-like Bap protein of Staphylococcus aureus as a tool to decorate the extracellular biofilm matrix or the bacterial cell surface with a battery of functional domains or proteins. We demonstrate that the localization of the functional tags can be change by simply modulating the pH of the medium. Using Bap features, we build a tool for trapping and covalent immobilizing molecules at bacterial cell surface or at the biofilm matrix based on the SpyTag/SpyCatcher system. Finally, we show that the cell wall of several Gram-positive bacteria could be functionalized through the external addition of the recombinant engineered Bap-amyloid domain. Overall, this work shows a simple and modulable system for biofilm functionalization based on the facultative protein Bap. © 2022, The Author(s).Publication Open Access Fluorescent molecular beacons mimicking RNA secondary structures to study RNA chaperone activity(Humana Press, 2020) Menéndez Gil, Pilar; Caballero Sánchez, Carlos; Solano Goñi, Cristina; Toledo Arana, Alejandro; Ciencias de la Salud; Osasun Zientziak; Universidad Pública de Navarra / Nafarroako Unibertsitate PublikoaMolecular beacons (MBs) are oligonucleotide probes with a hairpin-like structure that are typically labelled at the 5′ and 3′ ends with a fluorophore and a quencher dye, respectively. The conformation of the MB acts as a switch for fluorescence emission. When the fluorophore is in close proximity to the quencher, fluorescence emission cannot be detected, meaning that the switch is in an OFF state. However, if the MB structure is modified, separating the fluorophore from the quencher, the switch turns ON allowing fluorescence emission. This property has been extensively used for a wide variety of applications including real-time PCR reactions, study of protein-DNA interactions, and identification of conformational changes in RNA structures. Here, we describe a protocol based on the MB technology to measure the RNA unfolding capacities of the CspA RNA chaperone from Staphylococcus aureus. This method, with slight variations, may also be applied for testing the activity of other RNA chaperones, RNA helicases, or ribonucleases.Publication Open Access In vitro modeling of polyclonal infection dynamics within the human airways by Haemophilus influenzae differential fluorescent labeling(American Society for Microbiology, 2023) Rapún Araiz, Beatriz; Sorzabal-Bellido, Ioritz; Asensio López, Javier; Lázaro-Díez, María; Ariz Galilea, Mikel; Sobejano de la Merced, Carlos; Euba, Begoña; Fernández Calvet, Ariadna; Cortés Domínguez, Iván; Burgui Erice, Saioa; Toledo Arana, Alejandro; Ortiz de Solórzano, Carlos; Garmendia García, Juncal; Ingeniería Eléctrica, Electrónica y de Comunicación; Ingeniaritza Elektrikoa, Elektronikoa eta Telekomunikazio Ingeniaritza; Institute of Smart Cities - ISCStandardized clinical procedures for antibiotic administration rely on pathogen identification and antibiotic susceptibility testing, often performed on single-colony bacterial isolates. For respiratory pathogens, this could be questionable, as chronic patients may be persistently colonized by multiple clones or lineages from the same bacterial pathogen species. Indeed, multiple strains of nontypeable Haemophilus influenzae, with different antibiotic susceptibility profiles, can be co-isolated from cystic fibrosis and chronic obstructive pulmonary disease sputum specimens. Despite this clinical evidence, we lack information about the dynamics of H. influenzae polyclonal infections, which limits the optimization of therapeutics. Here, we present the engineering and validation of a plasmid toolkit (pTBH, toolbox for Haemophilus), with standardized modules consisting of six reporter genes for fluorescent or bioluminescent labeling of H. influenzae. This plasmid set was independently introduced in a panel of genomically and phenotypically different H. influenzae strains, and two of them were used as a proof of principle to analyze mixed biofilm growth architecture and antibiotic efficacy, and to visualize the dynamics of alveolar epithelial co-infection. The mixed biofilms showed a bilayer architecture, and antibiotic efficacy correlated with the antibiotic susceptibility of the respective single-species strains. Furthermore, differential kinetics of bacterial intracellular location within subcellular acidic compartments were quantified upon co-infection of cultured airway epithelial cells. Overall, we present a panel of novel plasmid tools and quantitative image analysis methods with the potential to be used in a whole range of bacterial host species, assay types, and¿or conditions and generate meaningful information for clinically relevant settings.Publication Open Access Anti-biofilm molecules targeting functional amyloids(MDPI, 2021) Matilla Cuenca, Leticia; Toledo Arana, Alejandro; Valle Turrillas, Jaione; Ciencias de la Salud; Osasun ZientziakThe choice of an effective therapeutic strategy in the treatment of biofilm-related infections is a significant issue. Amyloids, which have been historically related to human diseases, are now considered to be prevailing structural components of the biofilm matrix in a wide range of bacteria. This assumption creates the potential for an exciting research area, in which functional amyloids are considered to be attractive targets for drug development to dissemble biofilm structures. The present review describes the best-characterized bacterial functional amyloids and focuses on anti-biofilm agents that target intrinsic and facultative amyloids. This study provides a better understanding of the different modes of actions of the anti-amyloid molecules to inhibit biofilm formation. This information can be further exploited to improve the therapeutic strategies to combat biofilm-related infections.Publication Open Access Bacillus thuringiensis Cyt proteins as enablers of activity of Cry and Tpp toxins against Aedes albopictus(2023) Lai, Liliana; Villanueva, Maite; Muruzabal Galarza, Ane; Fernández González, Ana Beatriz; Unzue Pozas, Argiñe; Toledo Arana, Alejandro; Caballero Murillo, Primitivo; Caballero Sánchez, Carlos; Institute for Multidisciplinary Research in Applied Biology - IMABAedes albopictus is a species of mosquito, originally from Southeast Asia, that belongs to the Culicidae family and the Dipteran insect order. The distribution of this vector has rapidly changed over the past decade, making most of the temperate territories in the world vulnerable to important human vector-borne diseases such as dengue, yellow fever, zika or chikungunya. Bacillus thuringiensis var. israeliensis (Bti)-based insecticides represent a realistic alternative to the most common synthetic insecticides for the control of mosquito larvae. However, several studies have revealed emerging resistances to the major Bti Crystal proteins such as Cry4Aa, Cry4Ba and Cry11Aa, making the finding of new toxins necessary to diminish the exposure to the same toxicity factors overtime. Here, we characterized the individual activity of Cyt1Aa, Cry4Aa, Cry4Ba and Cry11Aa against A. albopictus and found a new protein, Cyt1A-like, that increases the activity of Cry11Aa more than 20-fold. Additionally, we demonstrated that Cyt1A-like facilitates the activity three new Bti toxins: Cry53-like, Cry56A-like and Tpp36-like. All in all, these results provide alternatives to the currently available Bti products for the control of mosquito populations and position Cyt proteins as enablers of activity for otherwise non-active crystal proteins.Publication Open Access Regulation of heterogenous lexA expression in staphylococcus aureus by an antisense RNA originating from transcriptional read-through upon natural mispairings in the sbrB intrinsic terminator(MDPI, 2022) Bastet, Laurène; Bustos-Sanmamed, Pilar; Catalán Moreno, Arancha; Caballero Sánchez, Carlos; Cuesta Ferre, Sergio; Matilla Cuenca, Leticia; Villanueva San Martín, Maite; Valle Turrillas, Jaione; Lasa Uzcudun, Íñigo; Toledo Arana, Alejandro; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako InstitutuaBacterial genomes are pervasively transcribed, generating a wide variety of antisense RNAs (asRNAs). Many of them originate from transcriptional read-through events (TREs) during the transcription termination process. Previous transcriptome analyses revealed that the lexA gene from Staphylococcus aureus, which encodes the main SOS response regulator, is affected by the presence of an asRNA. Here, we show that the lexA antisense RNA (lexA-asRNA) is generated by a TRE on the intrinsic terminator (TTsbrB) of the sbrB gene, which is located downstream of lexA, in the opposite strand. Transcriptional read-through occurs by a natural mutation that destabilizes the TTsbrB structure and modifies the efficiency of the intrinsic terminator. Restoring the mispairing mutation in the hairpin of TTsbrB prevented lexA-asRNA transcription. The level of lexA-asRNA directly correlated with cellular stress since the expressions of sbrB and lexA-asRNA depend on the stress transcription factor SigB. Comparative analyses revealed strain-specific nucleotide polymorphisms within TTsbrB, suggesting that this TT could be prone to accumulating natural mutations. A genome-wide analysis of TREs suggested that mispairings in TT hairpins might provide wider transcriptional connections with downstream genes and, ultimately, transcriptomic variability among S. aureus strains.