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Unlike the colicins, microcins and related peptide antibiotics, little is known about antibiotic proteins ([M.sub.r]> 10 000) from Gram-positive bacteria, since only few examples have been described to date. In this study we used heterologous expression of recombinant proteins to access the 17 kDa antibiotic protein SA-M57 from Streptococcus pyogenes, along with two proteins of unknown function identified in publicly available databases: EF1097 from Enterococcus faecalis and YpkK from Corynebacterium jeikeium. Here we show that all three are antibiotic proteins with different spectra of antimicrobial activity that kill sensitive bacteria at nanomolar concentrations. In silico structure predictions indicate that although the three proteins share little sequence similarity, they may be composed of conserved secondary structural elements: a relatively unstructured, acidic N-terminal portion and a basic C-terminal portion characterized by two helical elements separated by a loop structure and stabilized by an essential disulphide. Expression of individual segments as well as protein chimaeras revealed that, at least in the case of YpkK, the C-terminal portion is responsible for the killing action of the protein, whereas the role of the N-terminal portion remains unclear. Both scnM57 and ef1097 appear to be widely distributed in Strep. pyogenes and Ent. faecalis (respectively), whereas ypkK is found only rarely amongst clinical isolates of C. jeikeium. Finally, we determined that the proteins kill sensitive bacteria without lysis, a feature that distinguishes them from known murolytic proteins.

Staphylococcus aureus infections are becoming increasingly difficult to treat as they rapidly develop resistance to existing antibiotics. Bacterial type I signal peptidases are membrane-associated, cell-surface serine proteases with a unique catalytic mechanism that differs from that of eukaryotic endoplasmic reticulum signal peptidases. They are thus potential antimicrobial targets. S. aureus has a catalytically active type I signal peptidase, SpsB, that is essential for cell viability. To elucidate its structure, the spsB gene from S. aureus Newman strain was cloned and overexpressed in Escherichia coli. After exploring many different protein-modification constructs, SpsB was expressed as a fusion protein with maltose-binding protein and crystallized by hanging-drop vapour diffusion. The crystals belonged to the monoclinic space group P21 and diffracted to 2.05 Å resolution. The crystal structure of SpsB is anticipated to provide structural insight into Gram-positive signal peptidases and to aid in the development of antibacterial agents that target type I signal peptidases. [ABSTRACT FROM AUTHOR]

Epitope-specific immunotherapies have enabled the targeted treatment of a variety of diseases, ranging from cancer, infection, and autoimmune disorders. For CD8 + T cell-based therapies, the precise identification of immunogenic peptides presented by human leukocyte antigen (HLA) class I is essential which can be achieved by immunopeptidomics. Here, using lentivirus-mediated transduction and cell sorting approaches, we present a method to engineer a cell line that does not express its native HLA but instead expresses an HLA of interest (in this instance HLA-A*02:01). This technique can be used to elucidate the immunopeptidome of cell lines expressing different HLAs., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)