Human Acid Sphingomyelinase: An Important Drug Target with Versatile Roles in Staphylococcal Infection

Staphylococcus aureus is a Gram-positive human commensal, which asymptomatically colonizes around one third of the world population. Under certain circumstances, S. aureus can act as an opportunistic pathogen, causing diseases ranging from soft tissue and skin infections to severe life-threatening sepsis or endocarditis. Treatment of S. aureus patients becomes more and more challenging due to rising antibiotic resistances. In the last decades, it was recognized that S. aureus can invade and reside within host cells -an important strategy to evade the immune system and antibiotic treatment. In my thesis, I investigated the roles of sphingolipids and the lysosomal enzyme acid sphingomyelinase (ASM) in intracellular S. aureus infection. Thereby, I especially aimed to understand molecular mechanisms and how those processes could be targeted by pharmacological inhibition to prevent infection. In the first chapter, I characterized novel small molecule inhibitors of ASM. These compounds were based on ARC39, the most potent and selective ASM inhibitor known to date. Due to low membrane permeability, ARC39 possesses poor pharmacokinetics and thus, is not suitable for in vivo application. Here I demonstrate that protection of ARC39 with either acetoxymethyl (AM) or pivaloyloxymethyl (POM) groups enhanced the cellular uptake of the compound (kindly provided by Prof. Dr. C. Arenz, HU Berlin). Consequently, the inhibitor is rendered 100-200-fold more effective, as I showed by enzyme assays and cellular sphingolipid profiles. Furthermore, I found that POM groups were more stable (t\(1/2\)=~5h) than AM modifications (t\(1/2\)=~20 min) in blood serum, and hence, I suggest the POM-modified ARC39 derivative as a candidate for in vivo studies. This compound could be used for treatment of ASM-associated diseases, including S. aureus infection, in future. In the second part, I investigated the host cell response to one of the most important S. aureus virulence factors, the pore-forming α-toxin. We found that perforation of host cell plasma membranes through the toxin is accompanied by Ca\(²⁺\) influx and activation of the membrane repair enzyme mitogen-activated protein kinase (MAPK) p38. Consecutively, ASM is released by host cells for plasma membrane remodeling and endocytosis of membrane lesions to maintain cell integrity. In the third chapter, I demonstrated that ASM is enrolled in the internalization of S. aureus by host cells and characterized the underlying molecular mechanism. The bacteria presumably trigger nicotinic acid adenosine dinucleotide phosphate (NAADP)-dependent Ca\(²⁺\) mobilization from lysosomes, which results in lysosomal exocytosis and ASM release. The uptake of S. aureus requires ASM activity on the host cell surface, which likely leads to formation of lipid rafts and recruitment of neuronal cellular adhesion molecule (NRCAM) and protein tyrosine kinase 7 (PTK7), proteins that we identified via an ascorbate peroxidase 2 (APEX2)-based proximity screen. The ASM-mediated internalization pathway is very rapid and is responsible for cell entry, especially in early infection. Moreover, I showed that the route S. aureus uses to enter its host affects the intracellular fate of the pathogen, including phagosomal maturation and escape. In addition, we observed that sphingomyelin usually located on luminal membrane leaflets, is exposed to the host cytosol prior to phagosomal escape of S. aureus, a process that could allow host cells to sense of intra-phagosomal pathogens. Consistently, we identified several proteins that are recruited to cytosolically exposed SM via proximity labeling, which could be further investigated in future studies. In the last chapter, we established expansion microscopy (ExM) for S. aureus. ExM is a super resolution microscopy technique, in which the specimen is embedded into a swellable-hydrogel and then, physically is expanded. Hence, sample ultrastructure needs to be digested to enable isotropic expansion, which particularly was challenging due to the rigid and thick staphylococcal cell wall. Moreover, I developed staining protocols for novel trifunctional sphingomyelin analogues (kindly provided by Prof. Dr. J. Seibel, JMU Würzburg), which can be used to monitor sphingomyelinase activity by ExM.