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Quantitative proteomics unravels that the post-transcriptional regulator Crcmodulates the generation of vesicles and secreted virulence determinants of Pseudomonas aeruginosa

J Prot (2015), DOI: 10.1016/j.jprot.2015.06.009

Jose Antonio Reales-Calderón, Fernando Corona, Lucía Monteoliva, Concha Gil, Jose Luis Martínez.

Quantitative proteomics unravels that the post-transcriptional regulator Crc modulates the generation of vesicles and secreted virulence determinants of Pseudomonas aeruginosaRecent research indicates that the post-transcriptional regulator Crc modulates susceptibility to antibiotics and virulence in Pseudomonas aeruginosa. Several P. aeruginosa virulence factors are secreted or engulfed in vesicles. To decipher the Crc modulation of P. aeruginosa virulence, we constructed a crc deficient mutant and measure the proteome associated extracellular vesicles and the vesicle-free secretome using iTRAQ. Fifty vesicle-associated proteins were more abundant and 14 less abundant in the crc-defective strain, whereas 37 were more abundant and 17 less abundant in the vesicle-free secretome. Among them, virulence determinants, such as ToxA, protease IV, azurin, chitin-binding protein, PlcB and Hcp1, were less abundant in the crc-defective mutant. Transcriptomic analysis revealed that some of the observed changes were post-transcriptional and, thus, could be attributed to a direct Crc regulatory role; whereas, for other differentially secreted proteins, the regulatory role was likely indirect. We also observed that the crc mutant presented an impaired vesicle-associated secretion of quorum sensing signal molecules and less cytotoxicity than its wild-type strain. Our results offer new insights into the mechanisms by which Crc regulates P. aeruginosa virulence, through the modulation of vesicle formation and secretion of both virulence determinants and quorum sensing signals.


Referred to by:

Jose Antonio Reales-Calderón, Fernando Corona, Lucía Monteoliva, Concha Gil, Jose Luis Martínez. Quantitative proteomics unravels that the post-transcriptional regulator Crc modulates the generation of vesicles and secreted virulence determinants of Pseudomonas aeruginosaData in Brief, Volume 4, September 2015, Pages 450-453 doi:10.1016/j.dib.2015.07.002

Chemical Modification of a Dehydratase Enzyme Involved in Bacterial Virulence by an Ammonium Derivative: Evidence of its Active Site Covalent Adduct

J. Am. Chem. Soc. 2015; DOI:0.1021/jacs.5b04080

Concepción González-Bello, Lorena Tizón, Emilio Lence, José M. Otero, Mark J. van Raaij, Marta Martinez-Guitian, Alejandro Beceiro, Paul Thompson and Alastair R. Hawkins.

Chemical Modification of a Dehydratase Enzyme Involved in Bacterial Virulence by an Ammonium Derivative: Evidence of its Active Site Covalent AdductThe first example of an ammonium derivative that causes a specific modification of the active site of type I dehydroquinase (DHQ1), a dehydratase enzyme that is a promising target for antivirulence drug discovery, is described. The resolution at 1.35 Å of the crystal structure of DHQ1 from Salmonella typhi chemically modified by this ammonium derivative revealed that the ligand is covalently attached to the essential Lys170 through the formation of an amine. The detection by mass spectroscopy of the reaction intermediates, in conjunction with the results of molecular dynamics simulations, allowed us to explain the inhibition mechanism and the experimentally observed differences between S. typhi and Staphylococcus aureus enzymes. The results presented here reveal that the replacement of Phe225 in St-DHQ1 by Tyr214 in Sa-DHQ1 and its hydrogen bonding interaction with the conserved water molecule observed in several crystal structures protects the amino adduct against further dehydration/aromatization reactions. In contrast, for the St-DHQ1 enzyme, the carboxylate group of Asp114, with the assistance of this water molecule, would trigger the formation of a Schiff base that can undergo further dehydration reactions until full aromatization of the cyclohexane ring is achieved. Moreover, in vitro antivirulence studies showed that the reported compound is able to reduce the ability of Salmonella Enteritidis to kill A459 respiratory cells. These studies have identified a good scaffold for the design of irreversible inhibitors that can be used as drugs and has opened up new opportunities for the development of novel antivirulence agents by targeting the DHQ1 enzyme

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.

Transmission electron microscopy and the molecular structure of icosahedral viruses

Arch. Biochem. Biophys. 2015; doi:10.1016/j.abb.2015.06.001.

Carmen San Martín.

Transmission electron microscopy and the molecular structure of icosahedral virusesThe field of structural virology developed in parallel with methodological advances in X-ray crystallography and cryo-electron microscopy. At the end of the 1970s, crystallography yielded the first high resolution structure of an icosahedral virus, the T = 3 tomato bushy stunt virus at 2.9 Å. It took longer to reach near-atomic resolution in three-dimensional virus maps derived from electron microscopy data, but this was finally achieved, with the solution of complex icosahedral capsids such as the T = 25 human adenovirus at 3.5 Å. Both techniques now work hand-in-hand to determine those aspects of virus assembly and biology that remain unclear. This review examines the trajectory followed by EM imaging techniques in showing the molecular structure of icosahedral viruses, from the first two-dimensional negative staining images of capsids to the latest sophisticated techniques that provide high resolution three-dimensional data, or snapshots of the conformational changes necessary to complete the infectious cycle.

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.