A Asensio-Calavia, A Ceballos-Munuera, A Méndez-Pérez, B Álvarez, LA Fernández
Abstract
Biosafety of engineered bacteria as living therapeutics requires a tight regulation to control the specific delivery of protein effectors, maintaining minimum leakiness in the uninduced (OFF) state and efficient expression in the induced (ON) state. Here, we report a three repressors (3R) genetic circuit that tightly regulates the expression of multiple tac promoters (Ptac) integrated in the chromosome of E. coli and drives the expression of a complex type III secretion system injectisome for therapeutic protein delivery. The 3R genetic switch is based on the tetracycline repressor (TetR), the non-inducible lambda repressor cI (ind-) and a mutant lac repressor (LacIW220F) with higher activity. The 3R switch was optimized with different protein translation and degradation signals that control the levels of LacIW220F. We demonstrate the ability of an optimized switch to fully repress the strong leakiness of the Ptac promoters in the OFF state while triggering their efficient activation in the ON state with anhydrotetracycline (aTc), an inducer suitable for in vivo use. The implementation of the optimized 3R switch in the engineered synthetic injector E. coli (SIEC) strain boosts expression of injectisomes upon aTc induction, while maintaining a silent OFF state that preserves normal growth in the absence of the inducer. Since Ptac is a commonly used promoter, the 3R switch may have multiple applications for tight control of protein expression in E. coli. In addition, the modularity of the 3R switch may enable its tuning for the control of Ptac promoters with different inducers.
Science. 2021 Mar 12;371(6534):eabc9531.
David Ruano-Galleg, Julia Sanchez-Garrido, Zuzanna Kozik, Elena Núñez-Berrueco, Massiel Cepeda-Molero, Caroline Mullineaux-Sanders, Jasmine Naemi-Baghshomali Clark, Sabrina L Slater, Naama Wagne, Izabela Glegola-Madejska, Theodoros I Roumeliotis , Tal Pupko, Luis Ángel Fernández, Alfonso Rodríguez-Pató, Jyoti S Choudhary, Gad Frankel
Abstract
Infections with many Gram-negative pathogens, including Escherichia coli, Salmonella, Shigella, and Yersinia, rely on type III secretion system (T3SS) effectors. We hypothesized that while hijacking processes within mammalian cells, the effectors operate as a robust network that can tolerate substantial contractions. This was tested in vivo using the mouse pathogen Citrobacter rodentium (encoding 31 effectors). Sequential gene deletions showed that effector essentiality for infection was context dependent and that the network could tolerate 60% contraction while maintaining pathogenicity. Despite inducing very different colonic cytokine profiles (e.g., interleukin-22, interleukin-17, interferon-γ, or granulocyte-macrophage colony-stimulating factor), different networks induced protective immunity. Using data from >100 distinct mutant combinations, we built and trained a machine learning model able to predict colonization outcomes, which were confirmed experimentally. Furthermore, reproducing the human-restricted enteropathogenic E. coli effector repertoire in C. rodentium was not sufficient for efficient colonization, which implicates effector networks in host adaptation. These results unveil the extreme robustness of both T3SS effector networks and host responses.
doi: 10.1126/science.abc9531.
- Los resultados podrían ayudar a mejorar el diseño de vacunas y otros tratamientos frente a estos patógenos
- Los investigadores han combinado experimentos en el laboratorio con herramientas de inteligencia artificial
Un estudio en el que han participado científicos del Consejo Superior de Investigaciones Científicas (CSIC) ha logrado descifrar la forma en la que las bacterias patógenas, como Salmonella y Escherichia coli, usan redes de proteínas para controlar a las células durante una infección. Los resultados del trabajo, que han sido publicados en la revista Science, podrían ayudar a mejorar el diseño de vacunas y otros tratamientos frente a estos patógenos.
Muchas de las bacterias que producen enfermedades introducen proteínas maliciosas, denominadas efectores, en la célula, con las que reprograman sus funciones para beneficiar la infección. Los efectores toman el control de las células para, por ejemplo, evitar que se envíen señales de alarma al sistema inmune y así permitir a los intrusos colonizar el intestino.
“Los antibióticos son poco eficaces para combatir a estas infecciones ya que eliminan la flora bacteriana intestinal beneficiosa, la microbiota, y pueden acabar ayudando a las bacterias patógenas a colonizar el intestino. Por lo tanto, sigue siendo un reto encontrar tratamientos efectivos para estas infecciones y, para ello, es clave comprender cómo funciona el proceso infectivo”, explica el investigador del CSIC Luis Ángel Fernández, del Centro Nacional de Biotecnología (CNB-CSIC).
A study led by scientists from the Centro Nacional de Biotecnología of the CSIC finds fundamental genes for altering the intestinal mucosa. The research is a step toward development of an effective vaccine against Escherichia coli intestinal infections.
ACS Synth Biol. 2014; doi: 10.1021/sb500252a.
Piñero-Lambea C, Bodelón G, Fernández-Periáñez R, Cuesta AM, Alvarez-Vallina L, Fernández LA.
In this work we report synthetic adhesins (SAs) enabling the rational design of the adhesion properties of E. coli. SAs have a modular structure comprising a stable β-domain for outer membrane anchoring and surface-exposed immunoglobulin domains with high affinity and specificity that can be selected from large repertoires. SAs are constitutively and stably expressed in an E. coli strain lacking a conserved set of natural adhesins, directing a robust, fast, and specific adhesion of bacteria to target antigenic surfaces and cells.
We demonstrate the functionality of SAs in vivo, showing that, compared to wild type E. coli, lower doses of engineered E. coli are sufficient to colonize solid tumors expressing an antigen recognized by the SA. In addition, lower levels of engineered bacteria were found in non-target tissues. Therefore, SAs provide stable and specific adhesion capabilities to E. coli against target surfaces of interest for diverse applications using live bacteria.
Protein Expr Purif. 2013 Jul 12. pii: S1046-5928(13)00120-4
Salema V, Fernández LA.
Nanobodies (Nbs) are single domain antibodies based on the variable domains of heavy chain only antibodies (HCAbs) found in camelids, also referred to as VHHs. Their small size (ca. 12-15 kDa), superior biophysical and antigen binding properties have made Nbs very attractive molecules for multiple biotechnological applications, including human therapy. The most widely used system for the purification of Nbs is their expression in the periplasm of E. coli with a C-terminal hexa-histidine (His6) tag followed by immobilized metal affinity chromatography (IMAC). However, significant variability in the expression levels of different Nbs are routinely observed and a single affinity chromatography step is often not sufficient to obtain Nbs of high purity.
Here, we report an alternative method for expression and purification of Nbs from the periplasm of E. coli based on their fusion to maltose binding protein (MBP) in the N-terminus and His6 tag in the C-terminus (MBP-NbHis6). Soluble MBP-NbHis6 fusions were consistently expressed at high levels (⩾12 mg/L of induced culture in shake flasks) in the periplasm of E. coli HM140, a strain deficient in several periplasmic proteases. Highly pure MBP-NbHis6 fusions and free NbHis6 (after site specific proteolysis of the fusions), were recovered by amylose and metal affinity chromatography steps. The monomeric nature of the purified NbHis6 was determined by gel filtration chromatography. Lastly, we demonstrated by ELISA that both monomeric NbHis6 and MBP-NbHis6 fusions retained antigen binding activity and specificity, thus facilitating their direct use in antigen recognition assays.
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