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Background Increasing rates of antimicrobial resistance among uropathogens led, among other efforts, to the application of subtractive reverse vaccinology for the identification of antigens present in extraintestinal pathogenic E. coli (ExPEC) strains but absent or variable in non-pathogenic strains, in a quest for a broadly protective Escherichia coli vaccine. The protein coded by locus c5321 from CFT073 E. coli was identified as one of nine potential vaccine candidates against ExPEC and was able to confer protection with an efficacy of 33% in a mouse model of sepsis. c5321 (known also as EsiB) lacks functional annotation and structurally belongs to the Sel1-like repeat (SLR) family. Herein, as part of the general characterization of this potential antigen, we have focused on its structural properties. Results We report the 1.74 Å-resolution crystal structure of c5321 from CFT073 E. coli determined by Se-Met SAD phasing. The structure is composed of 11 SLR units in a topological organisation that highly resembles that found in HcpC from Helicobacter pylori, with the main difference residing in how the super-helical fold is stabilised. The stabilising effect of disulfide bridges in HcpC is replaced in c5321 by a strengthening of the inter-repeat hydrophobic core. A metal-ion binding site, uncharacteristic of SLR proteins, is detected between SLR units 3 and 4 in the region of the inter-repeat hydrophobic core. Crystal contacts are observed between the C-terminal tail of one molecule and the C-terminal amphipathic groove of a neighbouring one, resembling interactions between ligand and proteins containing tetratricopeptide-like repeats. Conclusions The structure of antigen c5321 presents a mode of stabilization of the SLR fold different from that observed in close homologs of known structure. The location of the metal-ion binding site and the observed crystal contacts suggest a potential role in regulation of conformational flexibility and interaction with yet unidentified target proteins, respectively. These findings open new perspectives in both antigen design and for the identification of a functional role for this protective antigen.

Staphylococcus aureus sortase A is a transpeptidase that is extensively used in various protein research applications. Sortase A is highly selective and does not require any cofactors for the catalysis of protein ligation and, importantly, can be produced in high yields. However, the primary disadvantage of this transpeptidase is its inability to access the recognition site within the highly structured regions of folded substrates. To overcome this problem, we developed an Escherichia coli expression system that produces milligram quantities of circularly closed sortase A; efficient enzyme cyclization was achieved by Synechocystis sp. PCC6803 intein-mediated post-translational splicing. The structural integrity of circular sortase A and its biochemical characteristics were compared to those of the linear enzyme analog and were found to be similar under native conditions. Additionally, the modified sortase was active at concentrations of urea up to 3 M and was capable of efficient catalytic protein–protein coupling, as shown by the ligation of purified glutathione-S-transferase and green fluorescence protein. In contrast to the circular enzyme, linear sortase A was unable to mediate the ligation of substrate proteins under the same conditions. Therefore, the proposed circular sortase A has improved enzymatic properties and has applications in advanced protein engineering and design.

A FRET-based random screening assay was used to generate hit compounds as sortase A inhibitors that allowed us to identify ethyl 3-oxo-2-(2-phenylhydrazinylidene)butanoate as an example of a new class of sortase A inhibitors. Other analogues were generated by changing the ethoxycarbonyl function for a carboxy, cyano or amide group, or introducing substituents in the phenyl ring of the ester and acid derivatives. The most active derivative found was 3-oxo-2-(2-(3,4dichlorophenyl)hydrazinylidene)butanoic acid (2b), showing an IC50 value of 50 µM. For a preliminary assessment of their antivirulence properties the new derivatives were tested for their antibiofilm activity. The most active compound resulted 2a, which showed inhibition of about 60% against S. aureus ATCC 29213, S. aureus ATCC 25923, S. aureus ATCC 6538 and S. epidermidis RP62A at a screening concentration of 100 µM.

There is an urgent need of anti-virulence agents effective in the prevention or eradication of biofilms that are intrinsically resistant to conventional antibiotics. If we consider that the first step of staphylococcal pathogenesis and of biofilm formation is the bacterial adhesion, promoted by the surface exposed proteins at the cell wall, we believe that new anti-virulence agents could be developed by using as a target the Sortase A (SrtA), the enzyme responsible of linking surface exposed proteins to peptidoglycan. Therefore, SrtA inhibitors could act as anti-adhesion agents useful to prevent Gram positive virulence mechanisms as well as a virulence mechanism based on biofilm formation. Starting from these consideration and in continuing our research work on antibacterial/antibiofilm agents, we thought it was of interest to obtain novel SrtA inhibitors, showing a molecular diversity than the known ones.The esters and the acids were tested for their inhibitory activity on SrtA, utilizing the FRET technics (Fluorescenze Resonance Energy Transfer) [1]. Some of them showed IC50 values in the range 50-100 μM. For a preliminary assessment of antivirulence properties the most active SrtA inhibitors were tested for their ability to interfere with biofilm formation of S. aureus ATCC 29213, S. aureus ATCC 25923, S.aureus ATCC 6538 and S. epidermidis RP62A. We found that the above derivatives interfere with biofilm formation

Gram-positive bacteria, in general, and staphylococci, in particular, are the widespread cause of nosocomial and community-acquired infections. The rapid evolvement of strains resistant to antibiotics currently in use is a serious challenge. Novel antimicrobial compounds have to be developed to fight these resistant bacteria, and sortase A, a bacterial cell wall enzyme, is a promising target for novel therapies. As a transpeptidase that covalently attaches various virulence factors to the cell surface, this enzyme plays a crucial role in the ability of bacteria to invade the host's tissues and to escape the immune response. In this study we have screened a small molecule library against recombinant Staphylococcus aureus sortase A using an in vitro FRET-based assay. The selected hits were validated by NMR methods in order to exclude false positives and to analyze the reversibility of inhibition. Further structural and functional analysis of the best hit allowed the identification of a novel class of benzisothiazolinone-based compounds as potent and promising sortase inhibitors.