Molecular and pre-clinical characterization of sphingosine-induced death of Pseudomonas aeruginosa

• Applicant: Professor Dr. Erich Gulbins
• Subject Area: Anatomy and Physiology ; Medical Microbiology and Mycology, Hygiene, Molecular Infection Biology
• Term: since 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 444075382

Pseudomonas aeruginosa is an important human pathogen, which could cause serious acute and chronic infections, especially in immunodeficient and cystic fibrosis (CF) patients, commonly in respiratory tract, the urinary tract, as wound infections or even sepsis. Respiratory infections with multiresistant P. aeruginosa are a major clinical problem. We have shown that sphingosine is abundantly expressed on epithelial cells of the respiratory tract in healthy humans and wildtype mice, while sphingosine is almost undetectable on the surface of epithelial cells of the respiratory tract from CF patients and CF mice. We have also previously demonstrated that sphingosine efficiently kills many bacterial species in vitro and in vivo, including P. aeruginosa, Staphylococcus aureus, Acinetobacter baumannii, Haemophilus influenzae, Escherichia coli, Burkholderia cepacia, Moraxella catarrhalis or Mycobacterium abscessus. In the previous grant period, we identified by biochemical and genetic studies cardiolipin as the primary target of sphingosine in P. aeruginosa. Our data revealed that sphingosine induces a degradation of cardiolipin by the Maintenance of Lipid Asymmetry (Mla)-system in P. aeruginosa, a system that serves the maintenance of the membrane asymmetry in gram-negative bacteria. Here, we aim to investigate whether (i) sphingosine binds to cardiolipin in the inner leaflet of the outer membrane and induces the translocation of cardiolipin into the outer leaflet followed by degradation of cardiolipin, (ii) sphingosine binds to LPS and thereby promotes the translocation of cardiolipin into the outer leaflet followed by its degradation (iii) sphingosine promotes degradation of cardiolipin by direct activation of MlaZ-transport and/or MlaY-lipase activity, (iv) sphingosine blocks other transporters (MlaA) that serve the recycling of cardiolipin and thereby shuttle the lipid to degradation, (v) sphingosine extracts cardiolipin directly from the outer and/or inner membrane and triggers degradation of cardiolipin and (vi) sphingosine reduces the binding of cardiolipin to proteins in the respiratory chain and thereby severely alters bacterial energy metabolism. Second, we will translate these data into preclinical models to develop sphingosine as novel antibacterial compound by testing whether (i) sphingosine kills multiresistent P. aeruginosa, (ii) is also active under hypoxic conditions, (iii) acts synergistically with polypeptide antibiotics by interfering with the bacterial outer membrane, (iv) and kills P. aeruginosa in the habitat as it is found in human CF and COPD lungs. Our studies aim to identify molecular mechanisms how sphingosine kills P. aeruginosa with a focus on the Mla-system in P. aeruginosa and attempt to transfer these studies to the pre-clinical development of sphingosine as a novel anti-bacterial drug.

• DFG Programme: Research Grants