Functional inhibition or genetic deletion of acid sphingomyelinase bacteriostatically inhibits Anaplasma phagocytophilum infection in vivo

Abstract

Anaplasma phagocytophilum infects neutrophils to cause granulocytic anaplasmosis. It poorly infects mice deficient in acid sphingomyelinase (ASM), a lysosomal enzyme critical for cholesterol efflux, and wild-type mice treated with desipramine that functionally inhibits ASM. Whether inhibition or genetic deletion of ASM is bacteriostatic or bactericidal for A. phagocytophilum and desipramine's ability to lower pathogen burden requires a competent immune system were unknown. A. phagocytophilum infected SCID mice were administered desipramine or PBS, followed by the transfer of blood to naïve wild-type mice. Next, infected wild-type mice were given desipramine or PBS followed by transfer of blood to naïve SCID mice. Finally, wild-type or ASM-deficient mice were infected and blood transferred to naïve SCID mice. The percentage of infected neutrophils was significantly reduced in all desipramine-treated or ASM-deficient mice and in all recipients of blood from these mice. Infection was markedly lower in ASM-deficient and desipramine-treated wild-type mice versus desipramine-treated SCID mice. Yet, infection was never ablated. Thus, ASM activity contributes to optimal A. phagocytophilum infection in vivo, pharmacologic inhibition or genetic deletion of ASM impairs infection in a bacteriostatic and reversible manner, and A. phagocytophilum is capable of co-opting ASM-independent lipid sources.

Acid Sphingomyelinase, a Lysosomal and Secretory Phospholipase C, Is Key for Cellular Phospholipid Catabolism

“…On the other hand, the functional inhibition of ASM activity stops infection cycles of vacuole adapted bacteria such as Anaplasma-phagocytophilum, Chlamydia trachomatis, and Chlamydia pneumoniae or even kills Coxiella burnettii [109,178]. ASM is also an essential regulator of mucosal immunity to enteric pathogens.…”

Ceramide-1-phosphate is a regulator of Golgi structure and is co-opted by the obligate intracellular bacterial pathogen Anaplasma phagocytophilum

“…Because ceramide generated by aSMase is a precursor of CERK-derived C1P ( Fig. 2A ), CERK is localized in the TGN ( 56 ), delivery of TGN-derived vesicles enriched in ceramide and sphingomyelin into the ApV is critical for A. phagocytophilum infection cycle progression ( 25 ), and disrupting ceramide generation by inhibiting aSMase halts the infection cycle ( 66 , 67 ), C1P levels were examined in infected versus uninfected cells using ultra high performance-liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS). Human promyelocytic HL-60 cells and primate RF/6A choroidal endothelial cells are well-established models for studying A. phagocytophilum -host interactions ( 25 , 43 , 44 , 66 , 70 – 73 ).…”

“…Importantly, CERK inhibitors are well tolerated in mice ( 50 ). As precedent for host-directed therapeutics against A. phagocytophilum , tricyclic antidepressants that inhibit aSMase, a key enzyme for the bacterium’s intra-MVB/TGN parasitic lifestyle, halt A. phagocytophilum infection ( 66 , 67 ). Future studies are needed to determine how C1P activates both Cdc42 and PKCα along with the mechanism by which A. phagocytophilum elevates CERK-derived C1P levels as little is known regarding CERK activation and sustained C1P levels in specific cellular topologies.…”

“…Three lines of evidence implicate C1P as a keystone factor for Golgi fragmentation and TGN parasitism during A. phagocytophilum infection. First, acid sphingomyelinase (aSMase), an MVB resident enzyme that converts sphingomyelin to ceramide ( 65 ), is critical for ApV expansion and maturation, RC-to-DC conversion, DC release from host cells, and is essential for A. phagocytophilum productive infection in mice ( 66 , 67 ). Second, ceramide kinase (CERK), which generates C1P via direct phosphorylation of aSMase-derived ceramide, localizes to the TGN ( 56 ).…”

Anaplasma phagocytophilum Hijacks Flotillin and NPC1 Complex To Acquire Intracellular Cholesterol for Proliferation, Which Can Be Inhibited with Ezetimibe

“…We previously showed U18886A and imipramine significantly inhibit A. phagocytophilum infection and replication in HL-60 cells in a dose-dependent manner ( 5 ). Recently, it was reported that desipramine, a metabolite of imipramine and an acid sphingomyelinase inhibitor, blocks LDL-derived cholesterol efflux, similar to U18886A or imipramine, and significantly inhibits A. phagocytophilum infection in cell culture and in mice ( 28 , 37 ). Taken together, these studies extend previous findings on the critical role of LDL-derived cholesterol for A. phagocytophilum proliferation ( 5 , 6 , 16 ); thus, LDL-derived cholesterol traffic can be a potential target of host-directed anti- A. phagocytophilum chemotherapy.…”