Conjugative relaxases as drivers of protein and DNA translocation through Type IV Secretion Systems: biological and biotechnological implications

Este trabajo se ha centrado en el estudio de diferentes aspectos de la actividad integrasa de las relaxasas conjugativas. Hemos analizado la relación entre la capacidad de oligomerizar de las relaxasas en presencia de su ADN diana y de catalizar la integración del ADN, utilizando como modelo la relaxasa TrwC. También, hemos estudiado el posible papel biológico de esta reacción en la colonización de huéspedes no permisivos. Además, hemos analizado la habilidad de la relaxasa MobA del plásmido RSF1010 en mediar la transferencia de ADN a una célula eucariota a través del SST4 del patógeno B. henselae, así como de promover la integración de este ADN en el genoma eucariota. Finalmente, hemos estudiado el potencial uso biotecnológico de las relaxasas conjugativas como sistemas de envío de ADN y proteínas, fusionando la relaxasa TrwC a la proteína Cas12a. Hemos comprobado que la proteína de fusión es activa y funcional en la célula receptora procariota, tras ser translocada a través del SST4.This work has focused on the study of different aspects of the integrase activity of conjugative relaxases. We have analyzed the relationship between the ability of relaxases to oligomerize in the presence of their target DNA and to catalyze DNA integration, using the TrwC relaxase as a model. Also, we have studied the possible biological role of this reaction in the colonization of non-permissive hosts. Furthermore, we have analyzed the ability of the MobA relaxase from plasmid RSF1010 to mediate the transfer of DNA to a eukaryotic cell through the T4SS of the pathogen B. henselae, as well as to promote the integration of this DNA into the eukaryotic genome. Finally, we have studied the potential biotechnological use of conjugative relaxases as DNA and protein delivery systems, fusing the TrwC relaxase to the Cas12a protein. We have verified that the fusion protein is active and functional in the prokaryotic receptor cell, after being translocated through T4SS.Esta Investigación ha sido financiada por una ayuda para contratos predoctorales en el área de la Biomedicina, Biotecnología y Ciencias de la Salud de la Universidad de Cantabria: 7665391046 Y0SC001170. El trabajo en el laboratorio de Matxalen Llosa Blas ha sido financiado por el Ministerio de Economía, Industria y Competitividad de España: BIO2013-46414-P y BIO2017-87190-R. Las estancias de investigación en el Instituto de Productos Lácteos de Asturias (IPLA) y en el Instituto Pasteur se realizaron gracias a sendas ayudas de la Universidad de Cantabria

The secret life of conjugative relaxases

Conjugative relaxases are well-characterized proteins responsible for the site- and strand-specific endonucleolytic cleavage and strand transfer reactions taking place at the start and end of the conjugative DNA transfer process. Most of the relaxases characterized biochemically and structurally belong to the HUH family of endonucleases. However, an increasing number of new families of relaxases are revealing a variety of protein folds and catalytic alternatives to accomplish conjugative DNA processing. Relaxases show high specificity for their cognate target DNA sequences, but several recent reports underscore the importance of their activity on secondary targets, leading to widespread mobilization of plasmids containing an oriT-like sequence. Some relaxases perform other functions associated with their nicking and strand transfer ability, such as catalyzing site-specific recombination or initiation of plasmid replication. They perform these roles in the absence of conjugation, and the validation of these functions in several systems strongly suggest that they are not mere artifactual laboratory observations. Other unexpected roles recently assigned to relaxases include controlling plasmid copy number and promoting retrotransposition. Their capacity to mediate promiscuous mobilization and genetic reorganizations can be exploited for a number of imaginative biotechnological applications. Overall, there is increasing evidence that conjugative relaxases are not only key enzymes for horizontal gene transfer, but may have been adapted to perform other roles which contribute to prokaryotic genetic plasticity. Relaxed target specificity may be key to this versatility.Acknowledgements: We are grateful to Mapi Garcillán-Barcia for helpful suggestions. Work in our lab is supported by grants BIO2017-87190-R from the MINECO (Spanish Ministry of Economy and Innovation), and IDEAS211LLOS from the AECC (Spanish Association Against Cancer) to ML. DLG-H is a recipient of a predoctoral appointment from the University of Cantabria

Conjugative DNA Transfer From E. coli to Transformation-Resistant Lactobacilli

Lactic acid bacteria (LAB) belonging to the genus classically known as Lactobacillus, recently split into 25 different genera, include many relevant species for the food industry. The well-known properties of lactobacilli as probiotics make them an attractive model also for vaccines and therapeutic proteins delivery in humans. However, scarce tools are available to accomplish genetic modification of these organisms, and most are only suitable for laboratory strains. Here, we test bacterial conjugation as a new tool to introduce genetic modifications into many biotechnologically relevant laboratory and wild type lactobacilli. Using mobilizable shuttle plasmids from a donor Escherichia coli carrying either RP4 or R388 conjugative systems, we were able to get transconjugants to all tested Lactocaseibacillus casei strains, including many natural isolates, and to several other genera, including Lentilactobacillus parabuchneri, for which no transformation protocol has been reported. Transconjugants were confirmed by the presence of the oriT and 16S rRNA gene sequencing. Serendipitously, we also found transconjugants into researcher-contaminant Staphylococcus epidermidis. Conjugative DNA transfer from E. coli to S. aureus was previously described, but at very low frequencies. We have purified this recipient strain and used it in standard conjugation assays, confirming that both R388 and RP4 conjugative systems mediate mobilization of plasmids into S. epidermidis. This protocol could be assayed to introduce DNA into other Gram-positive microorganisms which are resistant to transformation.FUNDING: Work in ML lab was supported by the grant BIO2017-87190-R from the Spanish Ministry of Science and Innovation. Work in MÁ lab was funded by the Spanish State Research Agency (AEI) and the European Regional Development Fund (FEDER) (AGL2016-78708-R, AEI/FEDER, EU). DG-H was a recipient of a predoctoral appointment from the University of Cantabria. RM-C received an ErasmusC traineeship grant

COVID-19 outbreaks in a transmission control scenario: challenges posed by social and leisure activities, and for workers in vulnerable conditions, Spain, early summer 2020

Severe acute respiratory syndrome coronavirus 2 community-wide transmission declined in Spain by early May 2020, being replaced by outbreaks and sporadic cases. From mid-June to 2 August, excluding single household outbreaks, 673 outbreaks were notified nationally, 551 active (>6,200 cases) at the time. More than half of these outbreaks and cases coincided with: (i) social (family/friends’ gatherings or leisure venues) and (ii) occupational (mainly involving workers in vulnerable conditions) settings. Control measures were accordingly applied

Proteína quimérica relaxasa–cas

ABSTRACT: The present invention relates to: the fusion protein relaxase-Cas12a; the CRISPR/Cas system that the fusion protein comprises; and the use of the relaxase-Cas12a fusion protein and/or CRISPR/Cas system to translocate to target cells, by means of the bacterial type IV secretion system, endonucleases and/or endonucleases bonded to DNA molecules, and to genetically modify the target cells.RESUMEN: La presente invención se refiere a la proteína de fusión Relaxasa-Cas12a, al sistema CRISPR/Cas que comprende dicha proteína de fusión y al uso de la proteína de fusión Relaxasa-Cas12a y/o del sistema CRISPR/Cas para la translocación de endonucleasas y/o endonucleasas unidas a moléculas de ADN, a células diana a través del sistema de secreción bacteriano tipo IV, y para la modificación genética de las células diana.Solicitud Internacional: PCT/ES2021/070626 (30.08.2021)Nº Pub. Solicitud Internacional: WO2022/043598A1 (03.03.2022

Proteína quimérica relaxasa-Cas

RESUMEN: La presente invención se refiere a la proteína de fusión Relaxasa-Cas12a, al sistema CRISPR/Cas que comprende dicha proteína de fusión y al uso de la proteína de fusión Relaxasa-Cas12a y/o del sistema CRISPR/Cas para la translocación de endonucleasas y/o endonucleasas unidas a moléculas de ADN, a células diana a través del sistema de secreción bacteriano tipo IV, y para la modificación genética de las células diana.Solicitud: 202030890 (28.08.2020)Nº Pub. de Solicitud: ES2897017A1 (28.02.2022

Recruitment of heterologous substrates by bacterial secretion systems for transkingdom translocation

Bacterial secretion systems mediate the selective exchange of macromolecules between bacteria and their environment, playing a pivotal role in processes such as horizontal gene transfer or virulence. Among the different families of secretion systems, Type III, IV and VI (T3SS, T4SS and T6SS) share the ability to inject their substrates into human cells, opening up the possibility of using them as customized injectors. For this to happen, it is necessary to understand how substrates are recruited and to be able to engineer secretion signals, so that the transmembrane machineries can recognize and translocate the desired substrates in place of their own. Other factors, such as recruiting proteins, chaperones, and the degree of unfolding required to cross through the secretion channel, may also affect transport. Advances in the knowledge of the secretion mechanism have allowed heterologous substrate engineering to accomplish translocation by T3SS, and to a lesser extent, T4SS and T6SS into human cells. In the case of T4SS, transport of nucleoprotein complexes adds a bonus to its biotechnological potential. Here, we review the current knowledge on substrate recognition by these secretion systems, the many examples of heterologous substrate translocation by engineering of secretion signals, and the current and future biotechnological and biomedical applications derived from this approach.Funding: This work was funded by grants PID2020-117956RB-I00 and PDC2021-120967-I00 from the Spanish Ministry of Science and Innovation (Agencia Estatal de Investigación) to ML. AF-G work was supported by a predoctoral contract from the University of Cantabria (Spain)