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Engineered bacteria as therapeutic agents

Curr Opin Biotechnol. 2015; 35: 94-102.

Piñero-Lambea C, Ruano-Gallego D, Fernández LÁ.

Curr Opin Biotechnol. 2015; 35: 94-102Although 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.

Engineering the controlled assembly of filamentous injectisomes in E. coli K-12 for protein translocation into mammalian cells

ACS Synth Biol. 2015 Jun 12.

Ruano-Gallego D, Álvarez B, Fernández LÁ.

ACS Synth Biol. 2015 Jun 12Bacterial 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.

Combined deletion of p38γ and p38δ reduces skin inflammation and protects from carcinogenesis

Oncotarget. 2015; 6(15): 12920-12935.

Zur R, Garcia-Ibanez L, Nunez-Buiza A, Aparicio N, Liappas G, Escós A, Risco A, Page A, Saiz-Ladera C, Alsina-Beauchamp D, Montans J, Paramio JM, Cuenda A.

Oncotarget. 2015; 6(15): 12920-12935The contribution of chronic skin inflammation to the development of squamous cell carcinoma (SCC) is poorly understood. While the mitogen-activated protein kinase p38α regulates inflammatory responses and tumour development, little is known about the role of p38γ and p38δ in these processes.

Here we show that combined p38γ and p38δ (p38γ/δ) deletion blocked skin tumour development in a chemically induced carcinogenesis model. p38γ/δ deletion reduced TPA-induced epidermal hyperproliferation and inflammation; it inhibited expression of proinflammatory cytokines and chemokines in keratinocytes in vitro and in whole skin in vivo, resulting in decreased neutrophil recruitment to skin. Our data indicate that p38γ/δ in keratinocytes promote carcinogenesis by enabling formation of a proinflammatory microenvironment that fosters epidermal hyperproliferation and tumourigenesis. These findings provide genetic evidence that p38γ and p38δ have essential roles in skin tumour development, and suggest that targeting inflammation through p38γ/δ offers a therapeutic strategy for SCC treatment and prevention.

The structure of the complex between α-tubulin, TBCE and TBCB reveals a tubulin dimer dissociation mechanism

J Cell Sci. 2015; 128(9): 1824-1834.

Serna M, Carranza G, Martín-Benito J, Janowski R, Canals A, Coll M, Zabala JC, Valpuesta JM.

J Cell Sci. 2015; 128(9): 1824-1834Tubulin proteostasis is regulated by a group of molecular chaperones termed tubulin cofactors (TBC). Whereas tubulin heterodimer formation is well‐characterized biochemically, its dissociation pathway is not clearly understood. Here, we carried out biochemical assays to dissect the role of the human TBCE and TBCB chaperones in α‐tubulin–β‐tubulin dissociation.

We used electron microscopy and image processing to determine the three‐dimensional structure of the human TBCE, TBCB and α‐tubulin (αEB) complex, which is formed upon α‐tubulin–β‐tubulin heterodimer dissociation by the two chaperones. Docking the atomic structures of domains of these proteins, including the TBCE UBL domain, as we determined by X‐ray crystallography, allowed description of the molecular architecture of the αEB complex. We found that heterodimer dissociation is an energy‐independent process that takes place through a disruption of the α‐tubulin–β‐tubulin interface that is caused by a steric interaction between β‐tubulin and the TBCE cytoskeleton‐associated protein glycine‐rich (CAP‐Gly) and leucine‐rich repeat (LRR) domains. The protruding arrangement of chaperone ubiquitin‐like (UBL) domains in the αEB complex suggests that there is a direct interaction of this complex with the proteasome, thus mediating α‐tubulin degradation.

Fish β-parvalbumin acquires allergenic properties by amyloid assembly

Swiss Med Wkly. 2015; 145: w14128.

Martínez J, Sánchez R, Castellanos M, Fernández-Escamilla AM, Vazquez-Cortés S, Fernández-Rivas M, Gasset M.

Swiss Med Wkly. 2015; 145: w14128PRINCIPLES: Amyloids are highly cross-β-sheet-rich aggregated states that confer protease resistance, membrane activity and multivalence properties to proteins, all essential features for the undesired preservation of food proteins transiting the gastrointestinal tract and causing type I allergy.

METHODS: Amyloid propensity of β-parvalbumin, the major fish allergen, was theoretically analysed and assayed under gastrointestinal-relevant conditions using the binding of thioflavin T, the formation of sodium dodecyl sulphate- (SDS-) resistant aggregates, circular dichroism spectroscopy and atomic force microscopy fibril imaging. Impact of amyloid aggregates on allergenicity was assessed with dot blot.

RESULTS: Sequences of β-parvalbumin from species with commercial value contain several adhesive hexapeptides capable of driving amyloid formation. Using Atlantic cod β-parvalbumin (rGad m 1) displaying high IgE cross-reactivity, we found that formation of amyloid fibres under simulated gastrointestinal conditions accounts for the resistance to acid and neutral proteases, for the presence of membrane active species under gastrointestinal relevant conditions and for the IgE-recognition in the sera of allergic patients. Incorporation of the anti-amyloid compound epigallocatechin gallate prevents rGad m 1 fibrillation, facilitates its protease digestion and impairs its recognition by IgE.

CONCLUSIONS: the formation of amyloid by rGad m 1 explains its degradation resistance, its facilitated passage across the intestinal epithelial barrier and its epitope architecture as allergen.