Graduation Year

2022

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Biology (Cell Biology, Microbiology, Molecular Biology)

Major Professor

Meera Nanjundan, Ph.D.

Committee Member

Charles E. Chalfant, Ph.D.

Committee Member

Michael N. Teng, Ph.D.

Committee Member

Sandy Westerheide, Ph.D.

Keywords

Cdc42, Ceramide kinase, ceramide-1-phosphate transport protein, Sphingolipids

Abstract

Anaplasma phagocytophilum, the etiologic agent of human granulocytic anaplasmosis (HGA), is a tick transmitted obligate intracellular bacterium that infects granulocytes, bone marrow progenitor cells and endothelial cells. HGA exhibits symptoms of febrile illness including fever, chills, headache, malaise, leukopenia and elevated liver enzymes which are treatable by antibiotics. However, elderly and immunocompromised patients are at a greater risk of developing fatal complications. Inside the host cell A.phagocytophilum resides in host cell derived vacuole called A.phagocytophilum occupied vacuole (ApV) where it undergoes its biphasic lifecycle. Being an intracellular pathogen, A.phagocytophilum relies heavily on nutrient acquisition from host cell and to obtain these nutrients they manipulate host Golgi-Endoplasmic reticulum (ER) vesicle trafficking system to reroute the nutrients to their ApV lumen. Since most obligate intracellular bacteria cannot synthesize their own sphingolipids, they uptake sphingolipids from host cell to promote their virulence and growth. Interestingly A.phagocytophilum is known to hijack ceramide rich trans Golgi (TGN) vesicles and inhibiting the delivery of these vesicles to the ApV lumen inhibits bacterial growth and impedes the generation of it infectious DC progeny.

Ceramide, the precursor of ceramide-1-phosphate (C1P), is at the center of sphingolipid biosynthetic pathway. A C1P, a potent regulator of cell proliferation, migration and invasion, and inflammatory cytokine production, is generated at the TGN through phosphorylation of ceramide by ceramide kinase (CERK). A C1P transport protein (CPTP) then transports the lipid to the plasma membrane or other organelles where it potentially undergoes a catabolic process. Therefore, inhibiting CPTP increases endogenous C1P levels and triggers Golgi fragmentation, a phenomenon that is also observed in A.phagocytophilum infected cells. However, the mechanism by which C1P induces Golgi fragmentation is not known. The role of C1P is well characterized in cancer biology, inflammatory diseases and wound healing; but little is known about its implication in microbial pathogenesis. Interestingly, Carlyon lab has shown that inhibiting ceramide generation by downregulation of acid sphingomyelinase (ASMase) arrests A.phagocytophilum growth both in in vitro and in vivo. Since TGN resident ceramide derived sphingolipids are crucial for A.phagocytophilum pathogenesis, hence we hypothesized that A.phagocytophilum elevates C1P levels in the TGN, increase in C1P induces Golgi fragmentation and disrupts Golgi-ER retrograde vesicle trafficking to promote anterograde traffic of TGN vesicles to ApV lumen and subsequently promote A.phagocytophilum growth. We have the following aims for this project 1) determine if CERK derived C1P is required for optimal A.phagocytophilum infection; 2) elucidate the mechanism by which C1P regulates Golgi-ER retrograde vesicle trafficking and Golgi fragmentation.

Here we show that A.phagocytophilum infected cells have higher C1P levels compared to non-infected cells and decreasing C1P levels by pharmacological inhibition and genetic ablation of CERK reduces A.phagocytophilum infection, disrupts bacterium’s biphasic lifecycle, and blocks A.phagocytophilum induced Golgi fragmentation. Conversely, increasing C1P levels reverses these effects and also facilitates ApV maturation. We further corroborated our result in vivo as CERK knockout mice (CERK -/-) were resistant to A.phagocytophilum infection. Our results also demonstrate that increasing C1P levels blocks Golgi-ER retrograde vesicle trafficking which is inversed by CERK inhibition. Our data further suggests that C1P stimulates Golgi fragmentation by inducing activation of protein kinase Cα (PKCα) followed by translocation of Cdc42 to the cellular membranes and its subsequent activation, which in turn activates c-Jun kinase (JNK). Consequently, PKCα and JNK triggers phosphorylation of Golgi restacking protein 55 (GRASP 55) and Golgi restacking protein 65 (GRASP 65) respectively, thereby inducing Golgi fragmentation.

Our project demonstrates the significance of CERK derived C1P in A.phagocytophilum pathogenesis and these findings recognize C1P as a potential target for non-antibiotic based treatment for A.phagocytophilum infection and also for other antibiotic resistant intracellular bacterial and other forms of microbial infections that depends on sphingolipid parasitism. Determining the role C1P in the maintenance of Golgi morphology lays the foundation to explore targeting CERK derived C1P in therapeutics of a plethora of diseases that is linked to Golgi fragmentation including Alzheimer’s, Huntington’s, Amyotrophic lateral sclerosis (AS) and various forms of cancer including lung, breast and pancreatic cancers.

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