In mammals, ceramide kinase (CerK)-mediated phosphorylation of ceramide is the only known pathway to ceramide-1-phosphate (C1P), a recently identified signaling sphingolipid metabolite. To help delineate the roles of CerK and C1P, we knocked out the gene of CerK in BALB/c mice by homologous recombination. All in vitro as well as cell-based assays indicated that CerK activity is completely abolished in Cerk−/− mice. Labeling with radioactive orthophosphate showed a profound reduction in the levels of de novo C1P formed in Cerk−/− macrophages. Consistently, mass spectrometry analysis revealed a major contribution of CerK to the formation of C16-C1P. However, the significant residual C1P levels in Cerk−/− animals indicate that alternative routes to C1P exist. Furthermore, serum levels of proapoptotic ceramide in these animals were significantly increased while levels of dihydroceramide as the biosynthetic precursor were reduced. Previous literature pointed to a role of CerK or C1P in innate immune cell function. Using a variety of mechanistic and disease models, as well as primary cells, we found that macrophage- and mast cell-dependent readouts are barely affected in the absence of CerK. However, the number of neutrophils was strikingly reduced in blood and spleen of Cerk−/− animals. When tested in a model of fulminant pneumonia, Cerk−/− animals developed a more severe disease, lending support to a defect in neutrophil homeostasis following CerK ablation. These results identify ceramide kinase as a key regulator of C1P, dihydroceramide and ceramide levels, with important implications for neutrophil homeostasis and innate immunity regulation.

LBM415 is an antibacterial agent belonging to the peptide deformylase inhibitor class of compounds. It has previously been shown to demonstrate good activity in vitro against a range of pathogens. In this study, the in vivo efficacy of LBM415 was evaluated in various mouse infection models. We investigated activity against a systemic infection model caused by intraperitoneal inoculation of Staphylococcus aureus (methicillin [ , a thigh infection model caused by intramuscular injection of MRSA, and a lung infection produced by intranasal inoculation of PSSP. In the systemic MSSA and MRSA infections, LBM415 was equivalent to linezolid and vancomycin. In the systemic PSSP infection, LBM415 was equivalent to linezolid, whereas against systemic MDRSP infection, the LBM415 50% effective dose (ED 50 ) was 4.8 mg/kg (dosed subcutaneously) and 36.6 mg/kg (dosed orally), compared to 13.2 mg/kg for telithromycin and >60 mg/kg for penicillin V and clarithromycin. In the MRSA thigh infection, LBM415 significantly reduced thigh bacterial levels compared to those of untreated mice, with levels similar to those after treatment with linezolid at the same dose levels. In the pneumonia model, the ED 50 to reduce the bacterial lung burden by >4 log 10 in 50% of treated animals was 23.3 mg/kg for LBM415, whereas moxifloxacin showed an ED 50 of 14.3 mg/kg. In summary, LBM415 showed in vivo efficacy in sepsis and specific organ infection models irrespective of resistance to other antibiotics. Results suggest the potential of peptide deformylase inhibitors as a novel class of therapeutic agents against antibiotic-resistant pathogens.With the emergence of pathogens resistant to current clinically used antibiotics, the need for new therapies has become of paramount importance. A novel class of antibacterial agents to emerge from research in this field is the peptide deformylase (PDF) inhibitors. PDF is a highly conserved metalloenzyme which deformylates the initial N-formyl methionine of newly synthesized bacterial polypeptides. This is an important step in bacterial protein synthesis, thus making it an attractive antibacterial target. The role of PDF and its attractiveness as an antibacterial target have previously been reviewed (10,11,15,16).LBM415 is one of the first compounds of the PDF inhibitor class to advance to clinical trials for the oral (p.o.) and parenteral treatment of respiratory tract and skin and skin structure infections caused by susceptible gram-positive and -negative organisms. LBM415 has been evaluated previously in vitro in comparison with other antibiotics and demonstrated potent activity against clinical strains of staphylococci, streptococci, enterococci, Moraxella catarrhalis, Legionella pneumophila, and Haemophilus influenzae (2,5,6,9,13,14). There was no difference in activity against strains classified as being susceptible or resistant to other classes of antibiotics. LBM415 also displayed activity against a collection of other gram-positive species, including Aerococcus spp., Bacillus spp., Corynebacterium ...

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Background S. aureus (SA) is a major human pathogen that causes invasive, clinical infections including bacteremia. Lefamulin (LEF) is the first semi-synthetic, pleuromutilin antibiotic for IV and oral use in humans. LEF is currently in Phase 3 trials for the treatment of community-acquired bacterial pneumonia (CABP). LEF specifically inhibits bacterial protein synthesis by binding to the peptidyl transferase center (PTC) via four H-bonds and other interactions at the A- and P-site resulting in an “induced fit.” LEF has been shown to be highly active against bacterial pathogens causing bacteremia, including SA. This study investigated the efficacy of LEF and comparators against SA in a neutropenic and immunocompetent murine bacteremia model.MethodsExperimentally induced MSSA bacteremia (inoculum ~2 × 107 CFU/mouse) was established in immunocompromised and immunocompetent mice. Infected mice received a single subcutaneous dose of either LEF or comparator (Table 1) 1 hours post-inoculation, mimicking human therapeutic exposures. A control group of infected mice were sacrificed directly before treatment to establish a baseline CFU count and comparison with the bacterial load of treated animals 24 hours post drug administration.ResultsIrrespective of the immune status, LEF showed superior efficacy to linezolid (LZD) and tigecycline (TGC) against MSSA, reducing the bacterial burden more than 4 log10 CFU/mL within 24 hours (Table 1). A comparable reduction of bacterial burden was observed between LEF and daptomycin (DAP) or vancomycin (VAN) treatment.ConclusionLEF showed comparable therapeutic outcome to DAP or VAN in this acute experimental infection model, while showing superior killing as compared with LZD or TGC. The efficacy of LEF was maintained under neutropenic conditions with >4log10 ΔCFU/mL at clinically relevant exposures. This study supports continued evaluation of LEF for as a potential treatment of staphylococcal bacteremia.Disclosures E. Fischer, Nabriva Therapeutics AG: Employee and Shareholder, Salary; B. C. Kappes, Nabriva Therapeutics AG: Employee and Shareholder, Salary; W. W. Wicha, Nabriva Therapeutics AG: Employee and Shareholder, Salary

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