Abstract The enterococcal cytolysin is a virulence factor consisting of two post-translationally modified peptides that synergistically kill human immune cells. Both peptides are made by CylM, a member of the LanM lanthipeptide synthetases. CylM catalyzes seven dehydrations of Ser and Thr residues and three cyclization reactions during the biosynthesis of the cytolysin large subunit. We present here the 2.2Å resolution structure of CylM, the first structural information on a LanM. Unexpectedly, the structure reveals that the dehydratase domain of CylM resembles the catalytic core of eukaryotic lipid kinases, despite the absence of clear sequence homology. The kinase and phosphate elimination active sites that affect net dehydration are immediately adjacent to each other. Characterization of mutants provided insights into the mechanism of the dehydration process. The structure is also of interest because of the interactions of human homologs of lanthipeptide cyclases with kinases such as mammalian target of rapamycin.

Enterococcus faecalis is a Gram-positive lactic acid intestinal opportunistic bacterium with virulence potential. For a better understanding of the adapation of this bacterium to the host conditions, we performed a transcriptome analysis of bacteria isolated from an infection site (mouse peritonitis) by RNA-sequencing. We identified a total of 211 genes with significantly higher transcript levels and 157 repressed genes. Our in vivo gene expression database reflects well the infection process since genes encoding important virulence factors like cy-tolysin, gelatinase or aggregation substance as well as stress response proteins, are signifi-cantly induced. Genes encoding metabolic activities are the second most abundant in vivo induced genes demonstrating that the bacteria are metabolically active and adapt to the special nutrient conditions of the host. α- and β- glucosides seem to be important substrates for E. faecalis inside the host. Compared to laboratory conditions, the flux through the upper part of glycolysis seems to be reduced and more carbon may enter the pentose phosphate pathway. This may reflect the need of the bacteria under infection conditions to produce more reducing power for biosynthesis. Another important substrate is certainly glycerol

Negligible in vivo growth of enterococci and high-level dispersion of data have led to inaccurate estimations of antibiotic phar-macodynamics (PD). Here we improved an in vivomodel apt for PD studies by optimizing the in vitro culture conditions for enterococci. The PD of vancomycin (VAN), ampicillin-sulbactam (SAM), and piperacillin-tazobactam (TZP) against enterococci were determined in vivo, comparing the following different conditions of inoculum preparation: aerobiosis, aerobiosis plus mu-cin, and anaerobiosis plus mucin. Drug exposure was expressed as the ratio of the area under the concentration-time curve for the free, unbound fraction of the drug to theMIC (fAUC/MIC) (VAN) or the time in a 24-h period that the drug concentration for the free, unbound fraction exceeded theMIC under steady-state pharmacokinetic conditions (fT>MIC) (SAM and TZP) and linked to the change in log10 CFU/thigh. Only anaerobiosis plus mucin enhanced the in vivo growth, yielding significant PD pa-rameters with all antibiotics. In conclusion, robust in vivo growth of enterococci was crucial for better determining the PD of tested antibacterial agents, and this was achieved by optimizing the procedure for preparing the inoculum. Enterococci are commensal organisms in the gastrointestinaltracts ofmany species, from insects to humans (1), but are also the third leading cause of hospital infections (2, 3). They display intrinsic and acquired resistance to almost all antibiotics in clini-cal use, and no single agent is able to kill more than 3 log10 CFU/g in vivo (4). Animal models of infection are invaluable tools for