Engineering Escherichia coli K-­12 for the secretion of single domain antibodies against attaching and effacing bacterial pathogens and for the injection of proteins of therapeutic potential into human cells

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 12-12-2014The attaching and effacing (A/E) bacterial pathogens infect the gastrointestinal tract of humans and other mammals after ingestion of contaminated food or water and cause persistent diarrhoea and other important diseases (e.g. haemolytic uremic syndrome, HUS) worldwide. Prototypical A/E pathogens are the enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) strains, which infect humans, as well as the mouse-­‐restricted pathogen Citrobacter rodentium (CR). These pathogens contain a common type III secretion system (T3SS): a macromolecular protein complex (the injectisome) assembled in the bacterial cell envelope that protrudes to the extracellular milieu with a filament of polymerized EspA. The T3SS allows translocation (injection) of a repertoire of bacterial proteins (called effectors) into the cytoplasm of the host enterocytes through the translocon subunits EspB and EspD, which insert in the host cell membrane. The effectors subvert multiple cellular functions and cause the effacement of the intestinal microvilli (A/E lesions) and the disruption of the intestinal epithelial barrier. Among them, the translocated intimin receptor (Tir) inserts in the host cell plasma membrane and is recognised by Intimin (Int), an outer membrane adhesin exposed on the bacterial cell surface. The Int:Tir interaction promotes the intimate attachment of the bacterium to the enterocyte and the polymerization of actin filament bundles (called “pedestals”) underneath the attached bacterium. Effective treatments to combat A/E pathogens are needed, since antibiotics activate the expression of the life-­‐threatening Shiga-­‐like toxins (Stx) from integrated pro-­‐phages, which are present in the more virulent strains -­‐ such as EHEC O157:H7. In this work we have assessed whether single domain antibodies (sdAbs) from camelids (also known as nanobodies or VHHs) binding essential proteins for A/E lesion formation (i.e., EspA, Int, Tir) could act as potential therapeutic agents against EHEC O157:H7 infections if secreted from non-­‐pathogenic E. coli strains. In addition, we have explored the biotechnological use of the filamentous T3SS of the A/E pathogens to inject VHHs and other proteins with therapeutic potential into the cytoplasm of human cells using a non-­‐ pathogenic E. coli K-­‐12 strain, engineered to express functional EPEC injectisomes. We have immunized a dromedary with purified EspA, Int280 and TirM (the protein domains involved in Int:Tir interaction) of EHEC to create a library of VHH genes. Selected VHHs clones from this library were secreted by commensal E. coli K-­‐12 strains carrying the α-­‐haemolysin (HlyA) secretion system and purified from the extracellular medium to characterise their binding and inhibitory properties of Int:Tir interaction in vitro. A high-­‐affinity VHH clone recognising TirM -­‐ named TD4 -­‐ and blocking Int:Tir interaction was found to interfere with the formation of actin pedestals on HeLa cells infected with EHEC. We have demonstrated that TD4 effectively competes with Int280 for the binding of Tir, since it has higher affinity towards TirM and recognises an epitope that overlaps with the necessary residues for the Int:Tir interaction. This VHH showed high specificity towards TirM of EHEC, not binding TirM of EPEC and only weakly with TirM of C. rodentium. With the aim to establish an in vivo mouse model for the evaluation of the A/E inhibition by TD4, we generated a C. rodentium strain expressing EHEC proteins Int, Tir, the multicargo chaperone CesT and the Tir-­‐coupling protein effector TccP. The resulting strain (CR-­‐EHEC) was capable of forming actin pedestals on HeLa cells, colonizing the mouse intestinal tract and inducing crypt hyperplasia, similarly to wild type C. rodentium. Importantly, we confirmed that TD4 also inhibits the formation of actin pedestals on HeLa cells induced by CR-­‐EHEC. These results leave open the possibility of testing the activity of TD4 against the formation of A/E lesions in vivo, which could represent the basis of a future EHEC infection treatment to control the outbreaks of this pathogen. In addition, we have addressed the biotechnological use of the filamentous T3SS of A/E pathogens for the injection of sdAbs and other heterologous proteins of therapeutic potential into the cytoplasm of human cells. The genes encoding the T3SS of EPEC are found in a 35 kb chromosomal island, called the Locus of Enterocyte Effacement (LEE). LEE is organized in various large operons (LEE1 to LEE5) and shorter transcriptional units (e.g. escD) and also contains genes encoding some effectors, their chaperones, transcriptional regulators, a muramidase and intimin. We aimed to engineer the expression of all known necessary genes for the assembly of a functional T3SS (27 genes in EPEC) in a non-­‐pathogenic E. coli strain (e.g. K-­‐12) under the control of the inducible promoter Ptac. We organised these genes in five engineered transcriptional units -­‐ operons eLEE1 to eLEE4 and eEscD -­‐ lacking effectors and transcriptional regulators. These engineered operons were sequentially integrated in specific sites of the genome of E. coli K-­‐12 corresponding with fimbrial and afimbrial adhesins. The resulting non-­‐pathogenic strain, referred to as Synthetic Injector E. coli (SIEC), was demonstrated to express the T3SS genes upon IPTG addition, to assemble functional injectisomes on its envelope and to be able to secrete the translocator proteins EspA, EspB, EspD. Interestingly, we found that the expression of the T3SS genes partially interfered with the assembly of the flagellum in E. coli K-­‐12, which can be explained considering the high identity between components of the T3SS and the flagellum. Lastly, we showed that a SIEC strain carrying an additional engineered operon encoding Int, Tir and CesT (eLEE5), was able to inject Tir to the cytoplasm of HeLa cells and to reproduce the intimate attachment of the bacterium to the host cell and the formation of actin pedestals. These results open the possible biomedical application of SIEC-­‐derived strains to inject proteins of therapeutic potential (e.g. VHHs, enzymes, transcription factors, toxins, effectors) against diverse human diseases (e.g. cancer, chronic inflammation)

A nanobody targeting the translocated intimin receptor inhibits the attachment of enterohemorrhagic E. coli to human colonic mucosa

Enterohemorrhagic E. coli (EHEC) is a human intestinal pathogen that causes hemorrhagic colitis and hemolytic uremic syndrome. No vaccines or specific therapies are currently available to prevent or treat these infections. EHEC tightly attaches to the intestinal epithelium by injecting the intimin receptor Tir into the host cell via a type III secretion system (T3SS). In this project, we identified a camelid single domain antibody (nanobody), named TD4, that recognizes a conserved Tir epitope overlapping the binding site of its natural ligand intimin with high affinity and stability. We show that TD4 inhibits attachment of EHEC to cultured human HeLa cells by preventing Tir clustering by intimin, activation of downstream actin polymerization and pedestal formation. Furthermore, we demonstrate that TD4 significantly reduces EHEC adherence to human colonic mucosa in in vitro organ cultures. Altogether, these results suggest that nanobody-based therapies hold potential in the development of much needed treatment and prevention strategies against EHEC infection

Targeted bacterial conjugation mediated by synthetic cell-to-cell adhesions

Genetic interventions on microbiomes, for clinical or biotechnological purposes, remain challenging. Conjugation-based delivery of genetic cargo is still unspecific and limited by low conjugation rates. Here we report an approach to overcome these problems, based on a synthetic bacterial adhesion system. Mating assemblers consist on a synthetic adhesion formed by the expression on the surface of donor and target cells of specific nanobodies (Nb) and their cognate antigen (Ag). The Nb?Ag bridge increased 1?3 logs transfer of a variety of plasmids, especially in liquid media, confirming that cell-cell docking is a main determinant limiting mating efficiency. Synthetic cell-to-cell adhesion allows efficient conjugation to targeted recipients, enhancing delivery of desired genes to a predefined subset of prey species, or even specific pathogenic strains such as enterohemorrhagic Escherichia coli (EHEC), within a bacterial community. The synthetic conjugation enhancer presented here optimizes plasmid delivery by selecting the target hosts with high selectivity.FUNDING: Spanish Science and Innovation Ministry (MCIN) [PID2020-117923GB-I00 to F.d.l.C.]; MCIN/AEI and FEDER [BIO2017-89081-R]; MCIN/AEI and NextGeneration EU/PRTR [PLEC2021-007739 to L.A.F.]. Conflict of interest statement. None declared ACKNOWLEDGEMENTS: We thank R. Fernandez-López for providing us with the BWmKate2 strain and, along with M. P. Garcillan-Barcia, for helpful discussions. We also thank E. Zechner for providing the pAR106 plasmid, and I. Rosenshine for EHEC strains and V. Campa for technical assistance in setting up the microscopy assay

Citrobacter rodentium Relies on Commensals for Colonization of the Colonic Mucosa.

We investigated the role of commensals at the peak of infection with the colonic mouse pathogen Citrobacter rodentium. Bioluminescent and kanamycin (Kan)-resistant C. rodentium persisted avirulently in the cecal lumen of mice continuously treated with Kan. A single Kan treatment was sufficient to displace C. rodentium from the colonic mucosa, a phenomenon not observed following treatment with vancomycin (Van) or metronidazole (Met). Kan, Van, and Met induce distinct dysbiosis, suggesting C. rodentium relies on specific commensals for colonic colonization. Expression of the master virulence regulator ler is induced in germ-free mice, yet C. rodentium is only seen in the cecal lumen. Moreover, in conventional mice, a single Kan treatment was sufficient to displace C. rodentium constitutively expressing Ler from the colonic mucosa. These results show that expression of virulence genes is not sufficient for colonization of the colonic mucosa and that commensals are essential for a physiological infection course

A922 Sequential measurement of 1 hour creatinine clearance (1-CRCL) in critically ill patients at risk of acute kidney injury (AKI)

Meeting abstrac

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Innovaciones y mejoras en el proyecto tutoría entre compañeros. Curso 2015-2016

Memoria ID-0137. Ayudas de la Universidad de Salamanca para la innovación docente, curso 2015-2016

Spatiotemporal Characteristics of the Largest HIV-1 CRF02_AG Outbreak in Spain: Evidence for Onward Transmissions

Background and Aim: The circulating recombinant form 02_AG (CRF02_AG) is the predominant clade among the human immunodeficiency virus type-1 (HIV-1) non-Bs with a prevalence of 5.97% (95% Confidence Interval-CI: 5.41–6.57%) across Spain. Our aim was to estimate the levels of regional clustering for CRF02_AG and the spatiotemporal characteristics of the largest CRF02_AG subepidemic in Spain.Methods: We studied 396 CRF02_AG sequences obtained from HIV-1 diagnosed patients during 2000–2014 from 10 autonomous communities of Spain. Phylogenetic analysis was performed on the 391 CRF02_AG sequences along with all globally sampled CRF02_AG sequences (N = 3,302) as references. Phylodynamic and phylogeographic analysis was performed to the largest CRF02_AG monophyletic cluster by a Bayesian method in BEAST v1.8.0 and by reconstructing ancestral states using the criterion of parsimony in Mesquite v3.4, respectively.Results: The HIV-1 CRF02_AG prevalence differed across Spanish autonomous communities we sampled from (p < 0.001). Phylogenetic analysis revealed that 52.7% of the CRF02_AG sequences formed 56 monophyletic clusters, with a range of 2–79 sequences. The CRF02_AG regional dispersal differed across Spain (p = 0.003), as suggested by monophyletic clustering. For the largest monophyletic cluster (subepidemic) (N = 79), 49.4% of the clustered sequences originated from Madrid, while most sequences (51.9%) had been obtained from men having sex with men (MSM). Molecular clock analysis suggested that the origin (tMRCA) of the CRF02_AG subepidemic was in 2002 (median estimate; 95% Highest Posterior Density-HPD interval: 1999–2004). Additionally, we found significant clustering within the CRF02_AG subepidemic according to the ethnic origin.Conclusion: CRF02_AG has been introduced as a result of multiple introductions in Spain, following regional dispersal in several cases. We showed that CRF02_AG transmissions were mostly due to regional dispersal in Spain. The hot-spot for the largest CRF02_AG regional subepidemic in Spain was in Madrid associated with MSM transmission risk group. The existence of subepidemics suggest that several spillovers occurred from Madrid to other areas. CRF02_AG sequences from Hispanics were clustered in a separate subclade suggesting no linkage between the local and Hispanic subepidemics

Immunoanalytical Detection of Conserved Peptides: Refining the Universe of Biomarker Targets in Planetary Exploration

Ancient peptides are remnants of early biochemistry that continue to play pivotal roles in current proteins. They are simple molecules yet complex enough to exhibit independent functions, being products of an evolved biochemistry at the interface of life and nonlife. Their adsorption to minerals may contribute to their stabilization and preservation over time. To investigate the feasibility of conserved peptide sequences and structures as target biomarkers for the search for life on Mars or other planetary bodies, we conducted a bioinformatics selection of well-conserved ancient peptides and produced polyclonal antibodies for their detection using fluorescence microarray immunoassays. Additionally, we explored how adsorbing peptides to Mars-representative minerals to form organomineral complexes could affect their immunological detection. The results demonstrated that the selected peptides exhibited autonomous folding, with some of them regaining their structure, even after denaturation. Furthermore, their cognate antibodies detected their conformational features regardless of amino acid sequences, thereby broadening the spectrum of target peptide sequences. While certain antibodies displayed unspecific binding to bare minerals, we validated that peptide–mineral complexes can be detected using sandwich immunoassays, as confirmed through desorption and competitive assays. Consequently, we conclude that the diversity of peptide sequences and structures suitable for use as target biomarkers in astrobiology can be constrained to a few well conserved sets, and they can be detected even if they are adsorbed in organomineral complexes