Design and construction of synthetic adhesins driving the specific attachment of Escherichia coli to target surfaces, cells, and tumors

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular: Fecha de lectura: 07-11-2014One of the aims of genetic engineering and synthetic biology is the design of microorganisms with novel capabilities that could be beneficial for humans, including their use of vaccines, diagnostic sensors, and therapeutic applications for major diseases such as cancer. In this regard, the availability of genetic elements able to program the adhesion of the engineered bacterium to different targets would be extremely useful. This work reports the development of synthetic adhesins (SAs) that enable to precisely program the adhesion properties of Escherichia coli bacteria to different target surfaces, including tumor cells. The structural organization of these novel SAs is defined by a domain that anchors the SA to the bacterial outer membrane, which is derived from the N-terminal fragment of Intimin comprising residues 1-659, and an adhesive domain based on the smallest antibody fragments known to date, termed VHHs. This modular organization allows the modification of the binding specificity of the SA by the exchange of the VHH sequence. We demonstrate that SAs are efficiently displayed on the surface of E. coli and are able to drive bacterial adhesion to antigen-coated abiotic surfaces and to target tumor cells expressing on their surface the antigen recognized by SAs. SAs are constitutively and stably expressing from the chromosome of an engineered E. coli strain lacking a conserved set of natural adhesins (i.e. type 1 fimbriae, Antigen 43 and mat fimbriae) and constitutively expressing the lux operon as bioluminescent reporter. Using tumor xenograft mouse models we have demonstrated that engineered E. coli strains carrying SAs colonize efficiently solid tumors expressing the cognate antigen recognized by the SA using two order of magnitude lower doses of systemically administered bacteria compared to control strains with SAs binding an unrelated antigen or the wild type E. coli strain. In addition, we observed that the engineered strains were cleared faster from non-target organs (e.g. liver and spleen) probably due to the deletion of natural adhesins. The fast and specific adhesion mediated by SAs was also employed to characterize the influence of both, flagella and YcgR protein in the adhesion process, as well as to investigate the short-term transcriptional response of E. coli upon adhesion to tumor cells. Our results indicate that, whereas active bacterial motility mediated by flagella is important for an efficient adhesion to target cells, the lack of YcgR protein does not affect the ability of bacteria to adhere to target cells, suggesting that the arrest of bacterial motility upon adhesion is independent of YcgR protein. In addition, we analyzed by RNAseq the global transcriptional response of E. coli bacteria upon adhesion (15 min) to target tumor cells. Our results indicate a common transcriptional response upon adhesion regardless of the cellular receptor recognized. Genes involved in sulfur uptake and its metabolism were upregulated upon adhesion, whereas genes involved in the transport of intermediates of the tricarboxylic acid cycle and tryptophan synthesis were downregulated. We found that the activity of gene fusions between the chromosomal yeeE promoter region and the mCherry reporter gene were upregulated in response to bacterial adhesion. Lastly, we have also demonstrated that SAs can drive the specific adhesion of non-live bacterial derived nanoparticles toward target tumor cells expressing a therapeutically relevant cell surface receptor (i.e. EGFR)

Engineered bacteria as therapeutic agents

Although bacteria are generally regarded as the causative agents of infectious diseases, most bacteria inhabiting the human body are non-pathogenic and some of them can be turned, after proper engineering, into ‘smart’ living therapeutics of defined properties for the treatment of different illnesses. This review focuses on recent developments to engineer bacteria for the treatment of diverse human pathologies, including inflammatory bowel diseases, autoimmune disorders, cancer, metabolic diseases and obesity, as well as to combat bacterial and viral infections. We discuss significant advances provided by synthetic biology to fully reprogram bacteria as human therapeutics, including novel measures for strict biocontainment.Ministerio de Economía y Competitividad (MINECO) (BIO2014- 60305R and BIO2011-26689), BACFITERed (SAF2014-56716-REDT)Comunidad Autónoma de Madrid (S2010-BMD-2312)La Caixa FoundationMINECO (BES-2009-02405)Peer reviewe

In Situ Functionalized Polymers for siRNA Delivery

NOTICE:This is the peer-reviewed version of the following article:Juan M. Priegue, Daniel N. Crisan, José Martínez-Costas, Juan R. Granja, Francisco Fernandez-Trillo, and Javier Montenegro (2016),“In situ Functionalized Polymers for siRNA Delivery ; Angew. Chem. Int. Ed., 55, 7492–7495 [doi: 10.1002/anie.201601441]. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for self-archivingA new method is reported herein for screening the biological activity of functional polymers across a consistent degree of polymerization and in situ, that is, under aqueous conditions and without purification/isolation of candidate polymers. In brief, the chemical functionality of a poly(acryloyl hydrazide) scaffold was activated under aqueous conditions using readily available aldehydes to obtain amphiphilic polymers. The transport activity of the resulting polymers can be evaluated in situ using model membranes and living cells without the need for tedious isolation and purification steps. This technology allowed the rapid identification of a supramolecular polymeric vector with excellent efficiency and reproducibility for the delivery of siRNA into human cells (HeLa-EGFP). The reported method constitutes a blueprint for the high-throughput screening and future discovery of new polymeric functional materials with important biological applicationsRoyal Society. Grant Number: IE130688 Spanish Ministry of Economy and Competitiveness. Grant Numbers: CTQ2014-59646-R, CTQ2013-43264-R, BFU2013-43513-R Birmingham Science City European Regional Development Fund Royal Society. Grant Number: RG140273 University of Birmingham MEC MINECO ERC. Grant Number: DYNAP-677786S

Engineering the Controlled Assembly of Filamentous Injectisomes in E. coli K-12 for Protein Translocation into Mammalian Cells.

Bacterial pathogens containing type III protein secretion systems (T3SS) assemble large needle-like protein complexes in the bacterial envelope, called injectisomes, for translocation of protein effectors into host cells. The application of these molecular syringes for the injection of proteins into mammalian cells is hindered by their structural and genomic complexity, requiring multiple polypeptides encoded along with effectors in various transcriptional units (TUs) with intricate regulation. In this work, we have rationally designed the controlled expression of the filamentous injectisomes found in enteropathogenic Escherichia coli (EPEC) in the nonpathogenic strain E. coli K-12. All structural components of EPEC injectisomes, encoded in a genomic island called the locus of enterocyte effacement (LEE), were engineered in five TUs (eLEEs) excluding effectors, promoters and transcriptional regulators. These eLEEs were placed under the control of the IPTG-inducible promoter Ptac and integrated into specific chromosomal sites of E. coli K-12 using a marker-less strategy. The resulting strain, named synthetic injector E. coli (SIEC), assembles filamentous injectisomes similar to those in EPEC. SIEC injectisomes form pores in the host plasma membrane and are able to translocate T3-substrate proteins (e.g., translocated intimin receptor, Tir) into the cytoplasm of HeLa cells reproducing the phenotypes of intimate attachment and polymerization of actin-pedestals elicited by EPEC bacteria. Hence, SIEC strain allows the controlled expression of functional filamentous injectisomes for efficient translocation of proteins with T3S-signals into mammalian cells

LoxTnSeq: random transposon insertions combined with cre/lox recombination and counterselection to generate large random genome reductions

The removal of unwanted genetic material is a key aspect in many synthetic biology efforts and often requires preliminary knowledge of which genomic regions are dispensable. Typically, these efforts are guided by transposon mutagenesis studies, coupled to deepsequencing (TnSeq) to identify insertion points and gene essentiality. However, epistatic interactions can cause unforeseen changes in essentiality after the deletion of a gene, leading to the redundancy of these essentiality maps. Here, we present LoxTnSeq, a new methodology to generate and catalogue libraries of genome reduction mutants. LoxTnSeq combines random integration of lox sites by transposon mutagenesis, and the generation of mutants via Cre recombinase, catalogued via deep sequencing. When LoxTnSeq was applied to the naturally genome reduced bacterium Mycoplasma pneumoniae, we obtained a mutant pool containing 285 unique deletions. These deletions spanned from > 50 bp to 28 Kb, which represents 21% of the total genome. LoxTnSeq also highlighted large regions of non-essential genes that could be removed simultaneously, and other non-essential regions that could not, providing a guide for future genome reductions.ISSN:1751-7915ISSN:1751-790

Tuning Gene Activity by Inducible and Targeted Regulation of Gene Expression in Minimal Bacterial Cells

Functional genomics studies in minimal mycoplasma cells enable unobstructed access to some of the most fundamental processes in biology. Conventional transposon bombardment and gene knockout approaches often fail to reveal functions of genes that are essential for viability, where lethality precludes phenotypic characterization. Conditional inactivation of genes is effective for characterizing functions central to cell growth and division, but tools are limited for this purpose in mycoplasmas. Here we demonstrate systems for inducible repression of gene expression based on clustered regularly interspaced short palindromic repeats-mediated interference (CRISPRi) in Mycoplasma pneumoniae and synthetic Mycoplasma mycoides, two organisms with reduced genomes actively used in systems biology studies. In the synthetic cell, we also demonstrate inducible gene expression for the first time. Time-course data suggest rapid kinetics and reversible engagement of CRISPRi. Targeting of six selected endogenous genes with this system results in lowered transcript levels or reduced growth rates that agree with lack or shortage of data in previous transposon bombardment studies, and now produces actual cells to analyze. The ksgA gene encodes a methylase that modifies 16S rRNA, rendering it vulnerable to inhibition by the antibiotic kasugamycin. Targeting the ksgA gene with CRISPRi removes the lethal effect of kasugamycin and enables cell growth, thereby establishing specific and effective gene modulation with our system. The facile methods for conditional gene activation and inactivation in mycoplasmas open the door to systematic dissection of genetic programs at the core of cellular life.This work was supported by internal funding from the J. Craig Venter Institute to H.O.S. and C.A.H., as well as grants BIO2013-4870R and BIO2013-50176EXP from the Ministerio de Economía y Competitividad to E.Q. and J.P., respectively, and Japan Society for the Promotion of Science KAKENHI grants JP26710015, JP15KK0266, and JP26106004 to S.K.A.M.M. is a recipient of a predoctoral fellowship from the Generalitat de Catalunya (FI-DGR 2014)

SynMyco transposon: engineering transposon vectors for efficient transformation of minimal genomes

Mycoplasmas are important model organisms for Systems and Synthetic Biology, and are pathogenic to a wide variety of species. Despite their relevance, many of the tools established for genome editing in other microorganisms are not available for Mycoplasmas. The Tn4001 transposon is the reference tool to work with these bacteria, but the transformation efficiencies (TEs) reported for the different species vary substantially. Here, we explore the mechanisms underlying these differences in four Mycoplasma species, Mycoplasma agalactiae, Mycoplasma feriruminatoris, Mycoplasma gallisepticum and Mycoplasma pneumoniae, selected for being representative members of each cluster of the Mycoplasma genus. We found that regulatory regions (RRs) driving the expression of the transposase and the antibiotic resistance marker have a major impact on the TEs. We then designed a synthetic RR termed SynMyco RR to control the expression of the key transposon vector elements. Using this synthetic RR, we were able to increase the TE for M. gallisepticum, M. feriruminatoris and M. agalactiae by 30-, 980- and 1036-fold, respectively. Finally, to illustrate the potential of this new transposon, we performed the first essentiality study in M. agalactiae, basing our study on more than 199,000 genome insertions.This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement 634942 (MycoSynVac) and was also financed by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme, under grant agreement 670216 (MYCOCHASSIS). We also acknowledge support of the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) to the EMBL partnership, the Centro de Excelencia Severo Ochoa and the CERCA Programme/Generalitat de Catalunya

LoxTnSeq: random transposon insertions combined with cre/lox recombination and counterselection to generate large random genome reductions

Data de publicació electrònica: 16-12-2020The removal of unwanted genetic material is a key aspect in many synthetic biology efforts and often requires preliminary knowledge of which genomic regions are dispensable. Typically, these efforts are guided by transposon mutagenesis studies, coupled to deepsequencing (TnSeq) to identify insertion points and gene essentiality. However, epistatic interactions can cause unforeseen changes in essentiality after the deletion of a gene, leading to the redundancy of these essentiality maps. Here, we present LoxTnSeq, a new methodology to generate and catalogue libraries of genome reduction mutants. LoxTnSeq combines random integration of lox sites by transposon mutagenesis, and the generation of mutants via Cre recombinase, catalogued via deep sequencing. When LoxTnSeq was applied to the naturally genome reduced bacterium Mycoplasma pneumoniae, we obtained a mutant pool containing 285 unique deletions. These deletions spanned from > 50 bp to 28 Kb, which represents 21% of the total genome. LoxTnSeq also highlighted large regions of non-essential genes that could be removed simultaneously, and other non-essential regions that could not, providing a guide for future genome reductions.This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement 634942 (MycoSynVac) and was also financed by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme, under grant agreement 670216 (MYCOCHASSIS). We also acknowledge support of the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) to the EMBL partnership, the Centro de Excelencia Severo Ochoa and the CERCA Programme/Generalitat de Cataluny

Inferring active metabolic pathways from proteomics and essentiality data

Here, we propose an approach to identify active metabolic pathways by integrating gene essentiality analysis and protein abundance. We use two bacterial species (Mycoplasma pneumoniae and Mycoplasma agalactiae) that share a high gene content similarity yet show significant metabolic differences. First, we build detailed metabolic maps of their carbon metabolism, the most striking difference being the absence of two key enzymes for glucose metabolism in M. agalactiae. We then determine carbon sources that allow growth in M. agalactiae, and we introduce glucose-dependent growth to show the functionality of its remaining glycolytic enzymes. By analyzing gene essentiality and performing quantitative proteomics, we can predict the active metabolic pathways connected to carbon metabolism and show significant differences in use and direction of key pathways despite sharing the large majority of genes. Gene essentiality combined with quantitative proteomics and metabolic maps can be used to determine activity and directionality of metabolic pathways.This project was financed by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program, under grant agreement nos. 634942 (MycoSynVac) and 670216 (MYCOCHASSIS). We acknowledge support of the Spanish Ministry of Science and Innovation to the EMBL partnership, the Centro de Excelencia Severo Ochoa, and the CERCA Programme / Generalitat de Catalunya

Mycoplasma pneumoniae genome editing based on oligo recombineering and Cas9-mediated counterselection

Mycoplasma species share a set of features, such as lack of a cell wall, streamlined genomes, simplified metabolism, and the use of a deviant genetic code, that make them attractive approximations of what a chassis strain should ideally be. Among them, Mycoplasma pneumoniae arises as a candidate for synthetic biology projects, as it is one of the most deeply characterized bacteria. However, the historical paucity of tools for editing Mycoplasma genomes has precluded the establishment of M. pneumoniae as a suitable chassis strain. Here, we developed an oligonucleotide recombineering method for this strain based on GP35, a ssDNA recombinase originally encoded by a Bacillus subtilis-associated phage. GP35-mediated oligo recombineering is able to carry out point mutations in the M. pneumoniae genome with an efficiency as high as 2.7 × 10-2, outperforming oligo recombineering protocols developed for other bacteria. Gene deletions of different sizes showed a decreasing power trend between efficiency and the scale of the attempted edition. However, the editing rates for all modifications increased when CRISPR/Cas9 was used to counterselect nonedited cells. This allowed edited clones carrying chromosomal deletions of up to 1.8 kb to be recovered with little to no screening of survivor cells. We envision this technology as a major step toward the use of M. pneumoniae, and possibly other Mycoplasmas, as synthetic biology chassis strains.This project has received funding from the European Union’s Horizon 2020 research and innovation program under Grant 634942 (MycoSynVac) and was also financed by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program under Grant 670216 (MYCOCHASSIS) and the FEDER project from Instituto Carlos III (ISCIII, Acción Estratégica en Salud 2016) (reference CP16/00094). We also acknowledge support of the Spanish Ministry of Science and Innovation, to the EMBL partnership, the Centro de Excelencia Severo Ochoa, and the CERCA Programme/Generalitat de Catalunya. Finally, we would like to thank Dr. Sarah A. Head for critical manuscript revision