Evidence for Induction of Integron-Based Antibiotic Resistance by the SOS Response in a Clinical Setting

Bacterial resistance to β-lactams may rely on acquired β-lactamases encoded by class 1 integron-borne genes. Rearrangement of integron cassette arrays is mediated by the integrase IntI1. It has been previously established that integrase expression can be activated by the SOS response in vitro, leading to speculation that this is an important clinical mechanism of acquiring resistance. Here we report the first in vivo evidence of the impact of SOS response activated by the antibiotic treatment given to a patient and its output in terms of resistance development. We identified a new mechanism of modulation of antibiotic resistance in integrons, based on the insertion of a genetic element, the gcuF1 cassette, upstream of the integron-borne cassette blaOXA-28 encoding an extended spectrum β-lactamase. This insertion creates the fused protein GCUF1-OXA-28 and modulates the transcription, the translation, and the secretion of the β-lactamase in a Pseudomonas aeruginosa isolate (S-Pae) susceptible to the third generation cephalosporin ceftazidime. We found that the metronidazole, not an anti-pseudomonal antibiotic given to the first patient infected with S-Pae, triggered the SOS response that subsequently activated the integrase IntI1 expression. This resulted in the rearrangement of the integron gene cassette array, through excision of the gcuF1 cassette, and the full expression the β-lactamase in an isolate (R-Pae) highly resistant to ceftazidime, which further spread to other patients within our hospital. Our results demonstrate that in human hosts, the antibiotic-induced SOS response in pathogens could play a pivotal role in adaptation process of the bacteria

Site-selective protein-modification chemistry for basic biology and drug development.

Nature has produced intricate machinery to covalently diversify the structure of proteins after their synthesis in the ribosome. In an attempt to mimic nature, chemists have developed a large set of reactions that enable post-expression modification of proteins at pre-determined sites. These reactions are now used to selectively install particular modifications on proteins for many biological and therapeutic applications. For example, they provide an opportunity to install post-translational modifications on proteins to determine their exact biological roles. Labelling of proteins in live cells with fluorescent dyes allows protein uptake and intracellular trafficking to be tracked and also enables physiological parameters to be measured optically. Through the conjugation of potent cytotoxicants to antibodies, novel anti-cancer drugs with improved efficacy and reduced side effects may be obtained. In this Perspective, we highlight the most exciting current and future applications of chemical site-selective protein modification and consider which hurdles still need to be overcome for more widespread use.We thank FCT Portugal (FCT Investigator to G.J.L.B.), the EU (Marie-Curie CIG to G.J.L.B. and Marie-Curie IEF to O.B.) and the EPSRC for funding. G.J.L.B. is a Royal Society University Research Fellow.This is the author accepted manuscript. The final version is available from NPG via http://dx.doi.org/10.1038/nchem.239

Supramolecular Architectures in 5, 5'-Substituted Hydantoins: Crystal Structures and Hirshfeld Surface Analyses

info:eu-repo/semantics/publishe

Native Chemical Ligation via N-Acylurea Thioester Surrogates Obtained by Fmoc Solid-Phase Peptide Synthesis

Native chemical ligation (NCL) enables the direct chemical synthesis and semisynthesis of proteins of different sizes and compositions, streamlining the access to proteins containing posttranslational modifications (PTMs). NCL assembles peptide fragments through the chemoselective reaction of a C-terminal α-thioester peptide, prepared either by chemical synthesis or via intein-splicing technology, and a recombinant or synthetic peptide containing an N-terminal Cys. Whereas the generation of C-terminal α-thioester proteins can be achieved via the recombinant fusion of the sequence of interest to an intein domain, chemical methods can also be used for synthetically accessible proteins. The use of Fmoc solid-phase peptide synthesis (Fmoc-SPPS) to obtain α-thioester peptides requires the development of novel strategies to overcome the lability of the thioester bond toward piperidine Fmoc-removal conditions. These new synthetic methods enable the easy introduction of PTMs in the thioester fragment. In this chapter, we describe an approach for the synthesis and use of C-terminal α-N-acylbenzimidazolinone (Nbz) and α-N-acyl-N′-methylbenzimidazolinone (MeNbz) peptides in NCL. Following stepwise peptide elongation, acylation with p-nitrophenylchloroformate and cyclization affords the Nbz/MeNbz peptides. The optimization of the coupling conditions allows the chemoselective incorporation of the C-terminal amino acid (aa) on the 3,4-diaminobenzoyl (Dbz) and prevents undesired diacylations of the resulting o-aminoanilide. Following synthesis, these Nbz/MeNbz peptides undergo NCL straightforwardly at neutral pH catalyzed by the presence of arylthiols. Herein, we apply the Nbz technology solid phase synthesis, NCL-mediated cyclization and folding of the heterodimeric RTD-1 defensin, an antimicrobial peptide isolated from the rhesus macaque leukocytes.This work was supported by the Spanish Ministerio de Economía y Competitividad (grants CTQ2012-31197 and RYC-2011-09001). J.P.-P. acknowledges an FPI scholarship (BES-2013-065237).Peer reviewe