Laura Fernández-García

Universidade da Coruña

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María Tomás

Instituto de Investigación Biomédica de A Coruña

Lucía Blasco

Instituto de Investigación Biomédica de A Coruña

María López

Hospital Universitario Infanta Sofía

Inés Bleriot

Instituto de Investigación Biomédica de A Coruña

Germán Bou

Complexo Hospitalario Universitario A Coruña

Olga Pacios

Instituto de Investigación Biomédica de A Coruña

Antón Ambroa

Complexo Hospitalario Universitario A Coruña

Felipe Fernández-Cuenca

Universidad de Sevilla

Thomas K. Wood

Pennsylvania State University

ÁP

Álvaro Pascual

Hospital Universitario Virgen Macarena

Cooperative Institutions

Instituto de Salud Carlos III

Centro de Investigación Biomédica en Red

Instituto de Investigación Biomédica de A Coruña

Complexo Hospitalario Universitario A Coruña

Red Espanola de Investigacion en Patologia Infecciosa

Universidade da Coruña

Fundación Seimc-Gesida

Universidad de Sevilla

Instituto de Biomedicina de Sevilla

Hospital Universitario Virgen Macarena

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Phage-Defense Systems Are Unlikely to Cause Cell Suicide

Viruses (2023)

Laura Fernández-García Thomas K. Wood

As new phage-defense systems (PDs) are discovered, the overlap between their mechanisms and those of toxin/antitoxin systems (TAs) is becoming clear in that both use similar means to reduce cellular metabolism; for example, both systems have members that deplete energetic compounds (e.g., NAD+, ATP) and deplete nucleic acids, and both have members that inflict membrane damage. Moreover, both TAs and PDs are similar in that rather than altruistically killing the host to limit phage propagation (commonly known as abortive infection), both reduce host metabolism since phages propagate less in slow-growing cells, and slow growth facilitates the interaction of multiple phage-defense systems.

Antitoxin

Nucleic acid metabolism

Cell metabolism

10.3390/v15091795

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Citations (11)

Hydrogels-Assisted Cell Engraftment for Repairing the Stroke-Damaged Brain: Chimera or Reality

Polymers (2018)

Daniel González‐Nieto Laura Fernández-García José Pérez‐Rigueiro Gustavo V. Guinea Fivos Panetsos

The use of advanced biomaterials as a structural and functional support for stem cells-based therapeutic implants has boosted the development of tissue engineering applications in multiple clinical fields. In relation to neurological disorders, we are still far from the clinical reality of restoring normal brain function in neurodegenerative diseases and cerebrovascular disorders. Hydrogel polymers show unique mechanical stiffness properties in the range of living soft tissues such as nervous tissue. Furthermore, the use of these polymers drastically enhances the engraftment of stem cells as well as their capacity to produce and deliver neuroprotective and neuroregenerative factors in the host tissue. Along this article, we review past and current trends in experimental and translational research to understand the opportunities, benefits, and types of tentative hydrogel-based applications for the treatment of cerebral disorders. Although the use of hydrogels for brain disorders has been restricted to the experimental area, the current level of knowledge anticipates an intense development of this field to reach clinics in forthcoming years.

Brain tissue

10.3390/polym10020184

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Citations (32)

Toxins of toxin/antitoxin systems are inactivated primarily through promoter mutations

Journal of Applied Microbiology (2019)

Laura Fernández-García Jun‐Seob Kim María Tomás Thomas K. Wood

Abstract Aims Given the extreme toxicity of some of the toxins of toxin-antitoxin (TA) systems, we were curious how the cell silences toxins, if the antitoxin is inactivated or independent toxins are obtained via horizontal gene transfer. Methods and Results Growth curves of Escherichia coli K12 BW25113 harbouring plasmid pCA24N to produce RalR, MqsR, GhoT or Hha toxins, showed toxin inactivation after 3 h. Sequencing plasmids from these cultures revealed toxin inactivation occurred primarily due to consistent deletions in the promoter. The lack of mutation in the structural genes was corroborated by a bioinformatics analysis of 1000 E. coli genomes which showed both conservation and little variability in the four toxin genes. For those strains that lacked a mutation in the plasmid, single nucleotide polymorphism analysis was performed to identify that chromosomal mutations iraM and mhpR inactivate the toxins GhoT and MqsR/GhoT respectively. Conclusion We find that the RalR (type I), MqsR (type II), GhoT (type V) and Hha (type VII) toxins are inactivated primarily by a mutation that inactivates the toxin promoter or via the chromosomal mutations iraM and mhpR. Significance and Impact of the Study This study demonstrates toxins of TA systems may be inactivated by mutations that primarily affect the toxin gene promoter instead of the toxin structural gene.

Antitoxin

Structural gene

10.1111/jam.14414

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Citations (10)

SAW Resonators and Filters Based on Sc0.43Al0.57N on Single Crystal and Polycrystalline Diamond

Micromachines (2022)

Miguel Sinusía Lozano Laura Fernández-García D. López‐Romero Oliver A. Williams G.F. Iriarte

The massive data transfer rates of nowadays mobile communication technologies demand devices not only with outstanding electric performances but with example stability in a wide range of conditions. Surface acoustic wave (SAW) devices provide a high Q-factor and properties inherent to the employed materials: thermal and chemical stability or low propagation losses. SAW resonators and filters based on Sc0.43Al0.57N synthetized by reactive magnetron sputtering on single crystal and polycrystalline diamond substrates were fabricated and evaluated. Our SAW resonators showed high electromechanical coupling coefficients for Rayleigh and Sezawa modes, propagating at 1.2 GHz and 2.3 GHz, respectively. Finally, SAW filters were fabricated on Sc0.43Al0.57N/diamond heterostructures, with working frequencies above 4.7 GHz and ~200 MHz bandwidths, confirming that these devices are promising candidates in developing 5G technology.

Thermal Stability

Crystal (programming language)

10.3390/mi13071061

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Citations (6)

Relationship Between Quorum Sensing and Secretion Systems

Frontiers in Microbiology (2019)

Rocío Trastoy Lucía Blasco Antón Ambroa Bertha González‐Pedrajo Laura Fernández-García

Quorum sensing (QS) is a communication mechanism between bacteria that allows specific processes to be controlled, such as biofilm formation, virulence factor expression, production of secondary metabolites and stress adaptation mechanisms such as bacterial competition systems including secretion systems (SS).These SS have an important role in bacterial communication.SS are ubiquitous; they are present in both Gram-negative and Gram-positive bacteria and in Mycobacterium sp.To date, 8 types of SS have been described (T1SS, T2SS, T3SS, T4SS, T5SS, T6SS, T7SS, and T9SS).They have global functions such as the transport of proteases, lipases, adhesins, heme-binding proteins, and amidases, and specific functions such as the synthesis of proteins in host cells, adaptation to the environment, the secretion of effectors to establish an infectious niche, transfer, absorption and release of DNA, translocation of effector proteins or DNA and autotransporter secretion.All of these functions can contribute to virulence and pathogenesis.In this review, we describe the known types of SS and discuss the ones that have been shown to be regulated by QS.Due to the large amount of information about this topic in some pathogens, we focus mainly on Pseudomonas aeruginosa and Vibrio spp.

10.3389/fmicb.2019.01100

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Citations (238)

CRISPR-Cas13a-Based Assay for Accurate Detection of OXA-48 and GES Carbapenemases

Microbiology Spectrum (2023)

Concha Ortiz-Cartagena Daniel Pablo‐Marcos Laura Fernández-García Lucía Blasco Olga Pacios

Carbapenem-resistant pathogens have been recognized as a health concern as they are both difficult to treat and detect in clinical microbiology laboratories. Researchers are making great efforts to develop highly specific, sensitive, accurate, and rapid diagnostic techniques, required to prevent the spread of these microorganisms and improve the prognosis of patients. In this context, CRISPR-Cas systems are proposed as promising tools for the development of diagnostic methods due to their high specificity; the Cas13a endonuclease can discriminate single nucleotide changes and displays collateral cleavage activity against single-stranded RNA molecules when activated. This technology is usually combined with isothermal pre-amplification reactions in order to increase its sensitivity. We have developed a new LAMP-CRISPR-Cas13a-based assay for the detection of OXA-48 and GES carbapenemases in clinical samples without the need for nucleic acid purification and concentration. To evaluate the assay, we used 68 OXA-48-like-producing Klebsiella pneumoniae clinical isolates as well as 64 Enterobacter cloacae complex GES-6, 14 Pseudomonas aeruginosa GES-5, 9 Serratia marcescens GES-6, 5 P. aeruginosa GES-6, and 3 P. aeruginosa (GES-15, GES-27, and GES-40) and 1 K. pneumoniae GES-2 isolates. The assay, which takes less than 2 h and costs approximately 10 € per reaction, exhibited 100% specificity and sensitivity (99% confidence interval [CI]) for both OXA-48 and all GES carbapenemases. IMPORTANCE Carbapenems are one of the last-resort antibiotics for defense against multidrug-resistant pathogens. Multiple nucleic acid amplification methods, including multiplex PCR, multiplex loop-mediated isothermal amplification (LAMP) and multiplex RPAs, can achieve rapid, accurate, and simultaneous detection of several resistance genes to carbapenems in a single reaction. However, these assays need thermal cycling steps and specialized instruments, giving them limited application in the field. In this work, we adapted with high specificity and sensitivity values, a new LAMP CRISPR-Cas13a-based assay for the detection of OXA-48 and GES carbapenemases in clinical samples without the need for RNA extraction.

Gram

10.1128/spectrum.01329-23

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Citations (9)

Genomic analysis of 40 prophages located in the genomes of 16 carbapenemase-producing clinical strains of Klebsiella pneumoniae

Microbial Genomics (2020)

Inés Bleriot Rocío Trastoy Lucía Blasco Felipe Fernández-Cuenca Antón Ambroa

Klebsiella pneumoniae is the clinically most important species within the genus Klebsiella and, as a result of the continuous emergence of multi-drug resistant (MDR) strains, the cause of severe nosocomial infections. The decline in the effectiveness of antibiotic treatments for infections caused by MDR bacteria has generated particular interest in the study of bacteriophages. In this study, we characterized a total of 40 temperate bacteriophages (prophages) with a genome range of 11.454-84.199 kb, predicted from 16 carbapenemase-producing clinical strains of K. pneumoniae belonging to different sequence types, previously identified by multilocus sequence typing. These prophages were grouped into the three families in the order Caudovirales (27 prophages belonging to the family Myoviridae, 10 prophages belonging to the family Siphoviridae and 3 prophages belonging to the family Podoviridae). Genomic comparison of the 40 prophage genomes led to the identification of four prophages isolated from different strains and of genome sizes of around 33.3, 36.1, 39.6 and 42.6 kb. These prophages showed sequence similarities (query cover >90 %, identity >99.9 %) with international Microbe Versus Phage (MVP) (http://mvp.medgenius.info/home) clusters 4762, 4901, 3499 and 4280, respectively. Phylogenetic analysis revealed the evolutionary proximity among the members of the four groups of the most frequently identified prophages in the bacterial genomes studied (33.3, 36.1, 39.6 and 42.6 kb), with bootstrap values of 100 %. This allowed the prophages to be classified into three clusters: A, B and C. Interestingly, these temperate bacteriophages did not infect the highest number of strains as indicated by a host-range assay, these results could be explained by the development of superinfection exclusion mechanisms. In addition, bioinformatic analysis of the 40 identified prophages revealed the presence of 2363 proteins. In total, 59.7 % of the proteins identified had a predicted function, mainly involving viral structure, transcription, replication and regulation (lysogenic/lysis). Interestingly, some proteins had putative functions associated with bacterial virulence (toxin expression and efflux pump regulators), phage defence profiles such as toxin-antitoxin modules, an anti-CRISPR/Cas9 protein, TerB protein (from terZABCDE operon) and methyltransferase proteins.

Prophage

Siphoviridae

Multilocus sequence typing

10.1099/mgen.0.000369

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Citations (33)

The role of PemIK (PemK/PemI) type II TA system from Klebsiella pneumoniae clinical strains in lytic phage infection

Scientific Reports (2022)

Inés Bleriot Lucía Blasco Olga Pacios Laura Fernández-García Antón Ambroa

Abstract Since their discovery, toxin-antitoxin (TA) systems have captivated the attention of many scientists. Recent studies have demonstrated that TA systems play a key role in phage inhibition. The aim of the present study was to investigate the role of the PemIK (PemK/PemI) type II TA system in phage inhibition by its intrinsic expression in clinical strains of Klebsiella pneumoniae carrying the lncL plasmid, which harbours the carbapenemase OXA-48 and the PemK/PemI TA system. Furthermore, induced expression of the system in an IPTG-inducible plasmid in a reference strain of K. pneumoniae ATCC10031 was also studied. The results showed that induced expression of the whole TA system did not inhibit phage infection, whereas overexpression of the pemK toxin prevented early infection. To investigate the molecular mechanism involved in the PemK toxin-mediated inhibition of phage infection, assays measuring metabolic activity and viability were performed, revealing that overexpression of the PemK toxin led to dormancy of the bacteria. Thus, we demonstrate that the PemK/PemI TA system plays a role in phage infection and that the action of the free toxin induces a dormant state in the cells, resulting in inhibition of phage infections.

Lytic cycle

Antitoxin

Lysogenic cycle

10.1038/s41598-022-08111-5

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Citations (27)

Evaluation of Neurosecretome from Mesenchymal Stem Cells Encapsulated in Silk Fibroin Hydrogels

Scientific Reports (2019)

Yolanda Martín-Martín Laura Fernández-García Miguel Hernández Sánchez-Rebato Núria Marí‐Buyé Francisco J. Rojo

Physical and cognitive disabilities are hallmarks of a variety of neurological diseases. Stem cell-based therapies are promising solutions to neuroprotect and repair the injured brain and overcome the limited capacity of the central nervous system to recover from damage. It is widely accepted that most benefits of different exogenously transplanted stem cells rely on the secretion of different factors and biomolecules that modulate inflammation, cell death and repair processes in the damaged host tissue. However, few cells survive in cerebral tissue after transplantation, diminishing the therapeutic efficacy. As general rule, cell encapsulation in natural and artificial polymers increases the in vivo engraftment of the transplanted cells. However, we have ignored the consequences of such encapsulation on the secretory activity of these cells. In this study, we investigated the biological compatibility between silk fibroin hydrogels and stem cells of mesenchymal origin, a cell population that has gained increasing attention and popularity in regenerative medicine. Although the survival of mesenchymal stem cells was not affected inside hydrogels, this biomaterial format caused adhesion and proliferation deficits and impaired secretion of several angiogenic, chemoattractant and neurogenic factors while concurrently potentiating the anti-inflammatory capacity of this cell population through a massive release of TGF-Beta-1. Our results set a milestone for the exploration of engineering polymers to modulate the secretory activity of stem cell-based therapies for neurological disorders.

Fibroin

Cell encapsulation

Regenerative Medicine

10.1038/s41598-019-45238-4

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Citations (37)

Genomic Analysis of Molecular Bacterial Mechanisms of Resistance to Phage Infection

Frontiers in Microbiology (2022)

Antón Ambroa Lucía Blasco María López Olga Pacios Inés Bleriot

To optimize phage therapy, we need to understand how bacteria evolve against phage attacks. One of the main problems of phage therapy is the appearance of bacterial resistance variants. The use of genomics to track antimicrobial resistance is increasingly developed and used in clinical laboratories. For that reason, it is important to consider, in an emerging future with phage therapy, to detect and avoid phage-resistant strains that can be overcome by the analysis of metadata provided by whole-genome sequencing. Here, we identified genes associated with phage resistance in 18 Acinetobacter baumannii clinical strains belonging to the ST-2 clonal complex during a decade (Ab2000 vs. 2010): 9 from 2000 to 9 from 2010. The presence of genes putatively associated with phage resistance was detected. Genes detected were associated with an abortive infection system, restriction–modification system, genes predicted to be associated with defense systems but with unknown function, and CRISPR-Cas system. Between 118 and 171 genes were found in the 18 clinical strains. On average, 26% of these genes were detected inside genomic islands in the 2000 strains and 32% in the 2010 strains. Furthermore, 38 potential CRISPR arrays in 17 of 18 of the strains were found, as well as 705 proteins associated with CRISPR-Cas systems. A moderately higher presence of these genes in the strains of 2010 in comparison with those of 2000 was found, especially those related to the restriction–modification system and CRISPR-Cas system. The presence of these genes in genomic islands at a higher rate in the strains of 2010 compared with those of 2000 was also detected. Whole-genome sequencing and bioinformatics could be powerful tools to avoid drawbacks when a personalized therapy is applied. In this study, it allows us to take care of the phage resistance in A. baumannii clinical strains to prevent a failure in possible phage therapy.

Phage therapy

Acinetobacter baumannii

Comparative Genomics

10.3389/fmicb.2021.784949

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Citations (22)