An organocatalyst-mediated protein aldol ligation (OPAL) affords C–C linked bioconjugates at neutral pH.

No abstract

Adhesive pili are external component of fibrous adhesive organelles and help bacteria attach to biotic or abiotic surfaces. The biogenesis of adhesive pili via the chaperone-usher pathway (CUP) is independent of external energy sources. In the classical CUP, chaperones transport assembly-competent pilins in a folded but expanded conformation. During donor-strand exchange, pilins subsequently collapse, producing a tightly packed hydrophobic core and releasing the necessary free energy to drive fiber formation. Here, we show that pilus biogenesis in non-classical, archaic, and alternative CUPs uses a different source of conformational energy. High-resolution structures of the archaic Csu-pili system from revealed that non-classical chaperones employ a short donor strand motif that is insufficient to fully complement the pilin fold. This results in chaperone-bound pilins being trapped in a substantially unfolded intermediate. The exchange of this short motif with the longer donor strand from adjacent pilin provides the full steric information essential for folding, and thereby induces a large unfolded-to-folded conformational transition to drive assembly. Our findings may inform the development of anti-adhesion drugs (pilicides) to combat bacterial infections.

Bacteria possess the ability to evolve varied and ingenious strategies to outwit the host immune system, instigating an evolutionary arms race. Proteases are amongst the many weapons employed by bacteria, which specifically cleave and neutralize key signalling molecules required for a coordinated immune response. In this article, we focus on a family of S8 subtilisin-like serine proteases expressed as cell-envelope proteases (CEPs) by group A and group B streptococci. Two of these proteases known as <i>Streptococcus pyogenes</i> CEP (SpyCEP) and C5a peptidase cleave the chemokine CXCL8 and the complement fragment C5a, respectively. Both CXCL8 and C5a are potent neutrophil-recruiting chemokines, and by neutralizing their activity, streptococci evade a key defence mechanism of innate immunity. We review the mechanisms by which CXCL8 and C5a recruit neutrophils and the characterization of SpyCEP and C5a peptidase, including both in vitro and in vivo studies. Recently described structural insights into the function of this CEP family are also discussed. We conclude by examining the progress of prototypic vaccines incorporating SpyCEP and C5a peptidase in their preparation. Since streptococci-producing SpyCEP and C5a peptidase are responsible for a considerable global disease burden, targeting these proteases by vaccination strategies or by small-molecule antagonists should provide protection from and promote the resolution of streptococcal infections.

Chemokines play diverse and fundamental roles in the immune system and human disease, which has prompted their structural and functional characterisation. Production of recombinant chemokines that are folded and bioactive is vital to their study but is limited by the stringent requirements of a native N-terminus for receptor activation and correct disulphide bonding required to stabilise the chemokine fold. Even when expressed as fusion proteins, overexpression of chemokines in E. coli tends to result in the formation of inclusion bodies, generating the additional steps of solubilisation and refolding. Here we present a novel method for producing soluble chemokines in relatively large amounts via a simple two-step purification procedure with no requirements for refolding. CXCL8 produced by this method has the correct chemokine fold as determined by NMR spectroscopy and in chemotaxis assays was indistinguishable from commercially available chemokines. We believe that this protocol significantly streamlines the generation of recombinant chemokines.