HUH site-specific recombinases for targeted modification of the human genome

Site-specific recombinases (SSRs) have been crucial in the development of mammalian transgenesis. For gene therapy purposes, this approach remains challenging, because, for example, SSR delivery is largely unresolved and SSR DNA substrates must pre-exist in target cells. In this review, we discuss the potential of His-hydrophobic-His (HUH) recombinases to overcome some of the limitations of conventional SSRs. Members of the HUH protein family cleave single-stranded (ss)DNA, but can mediate site-specific integration with the aid of the host replication machinery. Adeno-associated virus (AAV) Rep remains the only known example to support site-specific integration in human cells, and AAV is an excellent gene delivery vector that can be targeted to specific cells and organelles. Bacterial protein TrwC catalyzes integration into human sequences and can be delivered to human cells covalently linked to DNA, offering attractive new features for targeted genome modification.Work in our laboratories is supported by grant BIO2010-11623E from the Spanish Ministry of Science and Innovation to ML and UK Medical Research Council grant 1001764 to RML. CGP was a recipient of a predoctoral fellowship from the University of Cantabria (Spain).Peer Reviewe

The Conjugative Relaxase TrwC Promotes Integration of Foreign DNA in the Human Genome

Bacterial conjugation is a mechanism of horizontal DNA transfer. The relaxase TrwC of the conjugative plasmid R388 cleaves one strand of the transferred DNA at the oriT gene, covalently attaches to it, and leads the single-stranded DNA (ssDNA) into the recipient cell. In addition, TrwC catalyzes site-specific integration of the transferred DNA into its target sequence present in the genome of the recipient bacterium. Here, we report the analysis of the efficiency and specificity of the integrase activity of TrwC in human cells, using the type IV secretion system of the human pathogen Bartonella henselae to introduce relaxase-DNA complexes. Compared to Mob relaxase from plasmid pBGR1, we found that TrwC mediated a 10-fold increase in the rate of plasmid DNA transfer to human cells and a 100-fold increase in the rate of chromosomal integration of the transferred DNA. We used linear amplification-mediated PCR and plasmid rescue to characterize the integration pattern in the human genome. DNA sequence analysis revealed mostly reconstituted oriT sequences, indicating that TrwC is active and recircularizes transferred DNA in human cells. One TrwC-mediated site-specific integration event was detected, proving that TrwC is capable of mediating site-specific integration in the human genome, albeit with very low efficiency compared to the rate of random integration. Our results suggest that TrwC may stabilize the plasmid DNA molecules in the nucleus of the human cell, probably by recircularization of the transferred DNA strand. This stabilization would increase the opportunities for integration of the DNA by the host machinery. IMPORTANCE Different biotechnological applications, including gene therapy strategies, require permanent modification of target cells. Long-term expression is achieved either by extrachromosomal persistence or by integration of the introduced DNA. Here, we studied the utility of conjugative relaxase TrwC, a bacterial protein with site-specific integrase activity in bacteria, as an integrase in human cells. Although it is not efficient as a site-specific integrase, we found that TrwC is active in human cells and promotes random integration of the transferred DNA in the human genome, probably acting as a DNA chaperone until it is integrated by host mechanisms. TrwC-DNA complexes can be delivered to human cells through a type IV secretion system involved in pathogenesis. Thus, TrwC could be used in vivo to transfer the DNA of interest into the appropriate cell and promote its integration. If used in combination with a site-specific nuclease, it could lead to site-specific integration of the incoming DNA by homologous recombination

Selection of lactic acid bacteria as biopreservation agents and optimization of their mode of application for the control of Listeria monocytogenes in ready-to-eat cooked meat products

[EN] In order to meet consumers´demands for more natural foods and to find new methods to control foodborne pathogens in them, research is currently being focused on alternative preservation approaches, such as biopreservation with lactic acid bacteria (LAB). Here, a collection of lactic acid bacteria (LAB) isolates was characterized to identify potential biopreservative agents. Six isolates (one Lactococcus lactis, one Lacticaseibacillus paracasei and four Lactiplantibacillus plantarum) were selected based on their antimicrobial activity in in vitro assays. Whole genome sequencing showed that none of the six LAB isolates carried known virulence factors or acquired antimicrobial resistance genes, and that the L. lactis isolate was potentially a nisin Z producer. Growth of L. monocytogenes was successfully limited by L. lactis ULE383, L. paracasei ULE721 and L. plantarum ULE1599 throughout the shelf-life of cooked ham, meatloaf and roasted pork shoulder. These LAB isolates were also applied individually or as a cocktail at different inoculum concentrations (4, 6 and 8 log10 CFU/g) in challenge test studies involving cooked ham, showing a stronger anti-Listerial activity when a cocktail was used at 8 log10 CFU/g. Thus, a reduction of up to ~5.0 log10 CFU/g in L. monocytogenes growth potential was attained in cooked ham packaged under vacuum, modified atmosphere packaging or vacuum followed by high pressure processing (HPP). Only minor changes in color and texture were induced, although there was a significant acidification of the product when the LAB cultures were applied. Remarkably, this acidification was delayed when HPP was applied to the LAB inoculated batches. Metataxonomic analyses showed that the LAB cocktail was able to grow in the cooked ham and outcompete the indigenous microbiota, including spoilage microorganisms such as Brochothrix. Moreover, none of the batches were considered unacceptable in a sensory evaluation. Overall, this study shows the favourable antilisterial activity of the cocktail of LAB employed, with the combination of HPP and LAB achieving a complete inhibition of the pathogen with no detrimental effects in physico-chemical or sensorial evaluations, highlighting the usefulness of biopreservation approaches involving LAB for enhancing the safety of cooked meat products.S

Site-Specific Integration of Foreign DNA into Minimal Bacterial and Human Target Sequences Mediated by a Conjugative Relaxase

This is an open-access article distributed under the terms of the Creative Commons Attribution License.[Background]: Bacterial conjugation is a mechanism for horizontal DNA transfer between bacteria which requires cell to cell contact, usually mediated by self-transmissible plasmids. A protein known as relaxase is responsible for the processing of DNA during bacterial conjugation. TrwC, the relaxase of conjugative plasmid R388, is also able to catalyze site-specific integration of the transferred DNA into a copy of its target, the origin of transfer (oriT), present in a recipient plasmid. This reaction confers TrwC a high biotechnological potential as a tool for genomic engineering. [Methodology/Principal Findings]: We have characterized this reaction by conjugal mobilization of a suicide plasmid to a recipient cell with an oriT-containing plasmid, selecting for the cointegrates. Proteins TrwA and IHF enhanced integration frequency. TrwC could also catalyze integration when it is expressed from the recipient cell. Both Y18 and Y26 catalytic tyrosil residues were essential to perform the reaction, while TrwC DNA helicase activity was dispensable. The target DNA could be reduced to 17 bp encompassing TrwC nicking and binding sites. Two human genomic sequences resembling the 17 bp segment were accepted as targets for TrwC-mediated site-specific integration. TrwC could also integrate the incoming DNA molecule into an oriT copy present in the recipient chromosome. [Conclusions/Significance]: The results support a model for TrwC-mediated site-specific integration. This reaction may allow R388 to integrate into the genome of non-permissive hosts upon conjugative transfer. Also, the ability to act on target sequences present in the human genome underscores the biotechnological potential of conjugative relaxase TrwC as a site-specific integrase for genomic modification of human cells.This work was supported by grant BIO2008-00133 from the Spanish Ministry of Science and Innovation to ML. CGP was a recipient of a predoctoral fellowship from the University of Cantabria, Spain.Peer reviewe

Chloramphenicol Selection of IS10 Transposition in the cat Promoter Region of Widely Used Cloning Vectors

The widely used pSU8 family of cloning vectors is based on a p15A replicon and a chloramphenicol acetyltransferase (cat) gene conferring chloramphenicol resistance. We frequently observed an increase in the size of plasmids derived from these vectors. Analysis of the bigger molecular species shows that they have an IS10 copy inserted at a specific site between the promoter and the cat open reading frame. Promoter activity from both ends of IS10 has been reported, suggesting that the insertion events could lead to higher CAT production. Insertions were observed in certain constructions containing inserts that could lead to plasmid instability. To test the possibility that IS10 insertions were selected as a response to chloramphenicol selection, we have grown these constructs in the presence of different amounts of antibiotic and we observed that insertions arise promptly under higher chloramphenicol selective pressure. IS10 is present in many E. coli laboratory strains, so the possibility of insertion in constructions involving cat-containing vectors should be taken into account. Using lower chloramphenicol concentrations could solve this problem

Exposing and Overcoming Limitations of Clinical Laboratory Tests in COVID-19 by Adding Immunological Parameters; A Retrospective Cohort Analysis and Pilot Study

BackgroundTwo years since the onset of the COVID-19 pandemic no predictive algorithm has been generally adopted for clinical management and in most algorithms the contribution of laboratory variables is limited. ObjectivesTo measure the predictive performance of currently used clinical laboratory tests alone or combined with clinical variables and explore the predictive power of immunological tests adequate for clinical laboratories. Methods: Data from 2,600 COVID-19 patients of the first wave of the pandemic in the Barcelona area (exploratory cohort of 1,579, validation cohorts of 598 and 423 patients) including clinical parameters and laboratory tests were retrospectively collected. 28-day survival and maximal severity were the main outcomes considered in the multiparametric classical and machine learning statistical analysis. A pilot study was conducted in two subgroups (n=74 and n=41) measuring 17 cytokines and 27 lymphocyte phenotypes respectively. Findings1) Despite a strong association of clinical and laboratory variables with the outcomes in classical pairwise analysis, the contribution of laboratory tests to the combined prediction power was limited by redundancy. Laboratory variables reflected only two types of processes: inflammation and organ damage but none reflected the immune response, one major determinant of prognosis. 2) Eight of the thirty variables: age, comorbidity index, oxygen saturation to fraction of inspired oxygen ratio, neutrophil-lymphocyte ratio, C-reactive protein, aspartate aminotransferase/alanine aminotransferase ratio, fibrinogen, and glomerular filtration rate captured most of the combined statistical predictive power. 3) The interpretation of clinical and laboratory variables was moderately improved by grouping them in two categories i.e., inflammation related biomarkers and organ damage related biomarkers; Age and organ damage-related biomarker tests were the best predictors of survival, and inflammatory-related ones were the best predictors of severity. 4) The pilot study identified immunological tests (CXCL10, IL-6, IL-1RA and CCL2), that performed better than most currently used laboratory tests. ConclusionsLaboratory tests for clinical management of COVID 19 patients are valuable but limited predictors due to redundancy; this limitation could be overcome by adding immunological tests with independent predictive power. Understanding the limitations of tests in use would improve their interpretation and simplify clinical management but a systematic search for better immunological biomarkers is urgent and feasible

Site-specific recombination and integration reactions catalyzed by conjugative relaxases

Máster U. en Biología Molecular y Biomedicin

Evaluation of site-specific recombinase and integrase activity of conjugative relaxases in bacterial and human cell

ABSTRACT: With this work we have got insight into the molecular characterization of site-specific recombination and integration reactions mediated by relaxases. We have carried out an extensive mutagenesis of trwC that suggest that the improvement of relaxase TrwC as recombinase by altering one or two residues is not possible. Moreover, we have analysed the DNA requirements of the integration reaction by TrwC. Apart from TrwC, we have found that TraI proteins from plasmids F and pKM101 also have oriT-specific recombinase activity. In contrast, we did not detect F_TraI-mediated integration, suggesting a difference with TrwC. The potential use of TrwC for genomic engineering has been studied in human cells. We found that TrwC is active in human cells and it can act as a site-specific integrase, although not efficiently. The rate of integration of TrwC-driven DNA suggests that TrwC may be stabilizing the DNA in the human cell, favouring its random integration.RESUMEN: Con este trabajo hemos profundizado en la caracterización molecular de las reacciones de recombinación e integración sitio-específica mediadas por relaxasas. Hemos llevado a cabo una extensa mutagénesis de trwC que sugiere que su actividad recombinasa no se puede mejorar alterando uno o dos residuos. Además, hemos analizado los requerimientos de ADN de la reacción de integración mediada por TrwC. Aparte, hemos encontrado que las relaxasas de los plásmidos F y pKM101 también tienen actividad recombinasa oriT-específica. En cambio, no hemos detectado integración mediada por F_TraI, lo que sugiere diferencias con TrwC. Hemos estudiado el potencial uso de TrwC para ingeniería genética en células humanas. Encontramos que TrwC es activa en células humanas y que puede catalizar la integración sitio-específica, aunque no eficientemente. La tasa de integración de TrwC sugiere que la relaxasa podría estar estabilizando el ADN, favoreciendo su integración al azar

Improvement of recombination activity mediated by conjugative relaxases

DNA processing during bacterial conjugation is driven by a nucleoprotein complex called relaxosome, where the relaxase is the key protein. Relaxases are site-and strand-specific endonucleases which recognize and cleave one strand of their cognate origin of transfer (oriT), and are transferred to the recipient cell covalently bound to the 5‘ end of the nicked DNA strand. TrwC is the relaxase of the conjugative plasmid R388. TrwC is also a site-specific recombinase capable of promoting efficient recombination between two cognate oriTs in the absence of conjugation (César et al., 2006). Moreover, it can integrate the conjugatively transferred DNA strand into its target sequence present in the recipient cell (Draper et al., 2005). TrwC is able to act as a recombinase and integrase usign as targets a discrete number of sequences, located in noncoding sites of the human genome (Agúndez et al., 2011; Agúndez et al., 2012). TraI is the relaxase of the F plasmid, highly related to TrwC, although it has been reported that TraI does not have recombinase activity (César et al., 2006). Using a TraI-mediated site-specific recombination assay we have demonstrated that TraI can catalyze recombination between two F-oriT copies, although the frequency of recombination mediated by F-TraI is very low compared to that of TrwC under similar assay conditions. This allows future search for TraI mutants with increased recombination activity, which will provide additional target sequences in the genomes of higher organisms. We have used random mutagenic PCR to mutagenize the trwC and traI genes, selecting for mutants with enhanced recombination activity. We have obtained a TrwC mutant with increased recombination activity, which is completely functional in conjugation. We have tested its recombinase activity in the presence or absence of TrwA, its accessory protein. Furthemore, we have tested its activity as a site-specific integrase in a bacterial assay. Mutants with enhaced recombination activity could be used for genomic engineering with better efficiency than wild-type proteins. These and other results show that bacterial conjugative relaxases could be used in the future as site-specific integrases in human cells, leading the way for the development of a novel system for genomic modification which could have applications in biotechnology and biomedicine.Peer Reviewe

Site-specific recombination and integration reactions catalyzed by conjugative relaxases: a mutagenesis approach to improve recombination activity

Trabajo presentado a la: "IV International Conference on Environmental, Industrial and Applied Microbiology (BioMicroWorld2011)" celebrada del 14 al 16 de septiembre de 2011 en Torremolinos (España).Peer Reviewe