The Role of Lipid Droplets in Host-pathogen Interactions of Intracellular and Extracellular Bacteria

Context: Lipid droplets (LDs) are cytoplasmic lipid storage organelles that have recently gained importance in host-pathogen interactions. They are surrounded by a phospholipid monolayer and store excess cellular free fatty acids and cholesterol as triacylglycerol (TAG) and cholesterol ester (CE) respectively. LDs are crucial to lipid metabolism and energy homeostasis. Further, their role in membrane trafficking, cell signaling, and inflammation make them a prime target for pathogens. Several bacterial, viral, and protozoal pathogens exploit host- or self-derived LDs to build new infectious progeny and/or modulate inflammation to promote infection. However, the diverse role of LDs in different bacterial pathogens remains elusive and merits in-depth elucidation. Objective: The goal of this review is to summarize the current research describing how certain bacterial pathogens manipulate LDs, and how those LDs affect the survival and infectivity of said bacteria. Methods/Design: We investigated literature from the last ten years involving LD-pathogen interactions for the intracellular pathogens Mycobacterium tuberculosis, Mycobacterium leprae, and Mycobacterium bovis; and the extracellular pathogens Pseudomonas aeruginosa and Vibrio cholera. Results: Mycobacterium spp. use LDs as both a fatty acid reservoir and a source of immune mediators, thus enhancing bacterial growth. Pseudomonas mobilizes LDs to increase its virulence whereas V. cholera uses fatty acids derived from aquatic animals to remodel its membrane phospholipids and provide stability for the bacterium. Conclusion: Because LDs are important metabolic organelles, understanding how pathogens exploit LDs during infection will emphasize the underappreciated role of cellular metabolic processes during bacterial infections. Our review will shed light on the importance of LDs during intracellular and extracellular bacterial infections, exploring a novel aspect of host-pathogen interactions

Lipid Droplets Play an Important Role During Obligate Intracellular Bacterial Infections

Context: Lipid droplets (LDs) are cytoplasmic lipid storage organelles that have recently gained importance in host-pathogen interactions. Surrounded by a phospholipid monolayer, they store excess cellular free fatty acids and cholesterol as triacylglycerol and cholesterol ester, respectively. LDs are crucial to lipid metabolism, energy homeostasis, cell signaling, and inflammation, making them a prime target for pathogens. Several bacterial, viral, and protozoal pathogens exploit host- or self-derived LDs to build infectious progeny and/or modulate inflammation to promote infection. However, the diverse role of LDs in different bacterial pathogens remains elusive and merits in-depth elucidation. Objective: Our goal is to summarize the current research describing how certain bacterial pathogens manipulate LDs and how those LDs affect bacterial survival and infectivity. Methods/Design: We explored literature from the last ten years involving LD-pathogen interactions for the obligate intracellular pathogens Chlamydia trachomatis (Ct), Chlamydia pneumoniae (Cp), Coxiella burnetii (Cb), Anaplasma phagocytophilium (Ap), Ehrlichia chaffeensis (Ec), and Orientia tsutsugamushi (Ot). Results: Ct aggregates host LDs in the inclusion membrane which is speculated to be the bacterium’s nutrient source. Alternatively, there are no studies yet describing the relation between LDs and Cp,; however, this bacterium does show lipid dysregulation and production of pro-inflammatory cytokines during LD-rich foamy macrophage formation. Further, the tick-borne pathogens Ap and Ec use host-derived lipids for cell wall biosynthesis. Whereas, Ot and Cb manipulate LDs for survival. Conclusion: Our review sheds light on how obligate intracellular pathogens utilize LDs to promote survival in host cells. Since LDs are important metabolic organelles, focusing on their role will help us understand cellular metabolic processes important during host-pathogen interactions

Coxiella burnetii Blocks Intracellular Interleukin-17 Signaling in Macrophages

Coxiella burnetii is an obligate intracellular bacterium and the etiological agent of Q fever. Successful host cell infection requires the Coxiella type IVB secretion system (T4BSS), which translocates bacterial effector proteins across the vacuole membrane into the host cytoplasm, where they manipulate a variety of cell processes. To identify host cell targets of Coxiella T4BSS effector proteins, we determined the transcriptome of murine alveolar macrophages infected with a Coxiella T4BSS effector mutant. We identified a set of inflammatory genes that are significantly upregulated in T4BSS mutant-infected cells compared to mock-infected cells or cells infected with wild-type (WT) bacteria, suggesting that Coxiella T4BSS effector proteins downregulate the expression of these genes. In addition, the interleukin-17 (IL-17) signaling pathway was identified as one of the top pathways affected by the bacteria. While previous studies demonstrated that IL-17 plays a protective role against several pathogens, the role of IL-17 during Coxiella infection is unknown. We found that IL-17 kills intracellular Coxiella in a dose-dependent manner, with the T4BSS mutant exhibiting significantly more sensitivity to IL-17 than WT bacteria. In addition, quantitative PCR confirmed the increased expression of IL-17 downstream signaling genes in T4BSS mutant-infected cells compared to WT- or mock-infected cells, including the proinflammatory cytokine genes Il1a, Il1b, and Tnfa, the chemokine genes Cxcl2 and Ccl5, and the antimicrobial protein gene Lcn2 We further confirmed that the Coxiella T4BSS downregulates macrophage CXCL2/macrophage inflammatory protein 2 and CCL5/RANTES protein levels following IL-17 stimulation. Together, these data suggest that Coxiella downregulates IL-17 signaling in a T4BSS-dependent manner in order to escape the macrophage immune response

Contribution of Lipid Droplet Breakdown to Coxiella Burnetii Infection

Coxiella burnetii is an obligate intracellular bacterium and causative agent of culture-negative endocarditis. Although Coxiella initially infects alveolar macrophages, it is found in lipid droplet (LD)-containing foamy macrophages in endocarditis patients. LDs are host lipid storage organelles containing cholesterol esters (CE) and triacylglycerols (TAG). Our previous studies show that Coxiella actively manipulates host LD metabolism via its Type 4 Secretion System (T4SS), which secretes bacterial effectors in the host cell cytoplasm to manipulate cellular processes. Further, specifically blocking adipose triglyceride lipase (ATGL)-mediated LD breakdown inhibits Coxiella growth suggesting importance of LD-derived lipids for bacterial growth. However, how Coxiella regulates LD breakdown and the composition of LD-derived lipids is unknown. Our preliminary fluorescence microscopy studies using CRISPR knockouts and LD inhibitors indicate presence of TAG-rich LDs in Coxiella-infected cells. ATGL-mediated breakdown of TAG-rich LDs releases arachidonic acids, precursors for lipid immune mediators important for immunomodulation during bacterial infections. Hence we hypothesize that Coxiella manipulates ATGL via its T4SS to initiate TAG-rich LD breakdown and subsequently modulate the immune response to promote bacterial survival. To test this hypothesis, we analyzed ATGL gene expression in differentially infected cells using qRT-PCR. Compared to uninfected and T4SS-infected cells, Coxiella infection increased ATGL expression indicating T4SS-dependent regulation of ATGL. Ongoing studies are elucidating the Coxiella T4SS-ATGL interaction. To identify cellular CE and TAG levels and the breakdown products at different times post-infection, we are performing thin layer chromatography (TLC). Completion of our studies will identify the LD breakdown-derived lipids and how Coxiella regulates LD breakdown of to promote its intracellular survival

Manipulation of Host Cholesterol by Obligate Intracellular Bacteria

Cholesterol is a multifunctional lipid that plays important metabolic and structural roles in the eukaryotic cell. Despite having diverse lifestyles, the obligate intracellular bacterial pathogens Chlamydia, Coxiella, Anaplasma, Ehrlichia, and Rickettsia all target cholesterol during host cell colonization as a potential source of membrane, as well as a means to manipulate host cell signaling and trafficking. To promote host cell entry, these pathogens utilize cholesterol-rich microdomains known as lipid rafts, which serve as organizational and functional platforms for host signaling pathways involved in phagocytosis. Once a pathogen gains entrance to the intracellular space, it can manipulate host cholesterol trafficking pathways to access nutrient-rich vesicles or acquire membrane components for the bacteria or bacteria-containing vacuole. To acquire cholesterol, these pathogens specifically target host cholesterol metabolism, uptake, efflux, and storage. In this review, we examine the strategies obligate intracellular bacterial pathogens employ to manipulate cholesterol during host cell colonization. Understanding how obligate intracellular pathogens target and use host cholesterol provides critical insight into the host-pathogen relationship

Elevated Cholesterol in the Coxiella burnetii Intracellular Niche Is Bacteriolytic

Coxiella burnetii is an intracellular bacterial pathogen and a significant cause of culture-negative endocarditis in the United States. Upon infection, the nascent Coxiella phagosome fuses with the host endocytic pathway to form a large lysosome-like vacuole called the parasitophorous vacuole (PV). The PV membrane is rich in sterols, and drugs perturbing host cell cholesterol homeostasis inhibit PV formation and bacterial growth. Using cholesterol supplementation of a cholesterol-free cell model system, we found smaller PVs and reduced Coxiella growth as cellular cholesterol concentration increased. Further, we observed in cells with cholesterol a significant number of nonfusogenic PVs that contained degraded bacteria, a phenotype not observed in cholesterol-free cells. Cholesterol had no effect on axenic Coxiella cultures, indicating that only intracellular bacteria are sensitive to cholesterol. Live-cell microscopy revealed that both plasma membrane-derived cholesterol and the exogenous cholesterol carrier protein low-density lipoprotein (LDL) traffic to the PV. To test the possibility that increasing PV cholesterol levels affects bacterial survival, infected cells were treated with U18666A, a drug that traps cholesterol in lysosomes and PVs. U18666A treatment led to PVs containing degraded bacteria and a significant loss in bacterial viability. The PV pH was significantly more acidic in cells with cholesterol or cells treated with U18666A, and the vacuolar ATPase inhibitor bafilomycin blocked cholesterol-induced PV acidification and bacterial death. Additionally, treatment of infected HeLa cells with several FDA-approved cholesterol-altering drugs led to a loss of bacterial viability, a phenotype also rescued by bafilomycin. Collectively, these data suggest that increasing PV cholesterol further acidifies the PV, leading to Coxiella death.IMPORTANCE The intracellular Gram-negative bacterium Coxiella burnetii is a significant cause of culture-negative infectious endocarditis, which can be fatal if untreated. The existing treatment strategy requires prolonged antibiotic treatment, with a 10-year mortality rate of 19% in treated patients. Therefore, new clinical therapies are needed and can be achieved by better understanding C. burnetii pathogenesis. Upon infection of host cells, C. burnetii grows within a specialized replication niche, the parasitophorous vacuole (PV). Recent data have linked cholesterol to intracellular C. burnetii growth and PV formation, leading us to further decipher the role of cholesterol during C. burnetii-host interaction. We observed that increasing PV cholesterol concentration leads to increased acidification of the PV and bacterial death. Further, treatment with FDA-approved drugs that alter host cholesterol homeostasis also killed C. burnetii through PV acidification. Our findings suggest that targeting host cholesterol metabolism might prove clinically efficacious in controlling C. burnetii infection

Role of cyclooxygenase inhibitors in blocking Coxiella burnetii intracellular growth.

Coxiella burnetii is an obligate intracellular bacterial pathogen and a causative agent of culture-negative endocarditis. While Coxiella initially infects alveolar macrophages, it may disseminate and cause endocarditis up to 20 years after initial infection. If untreated, Coxiella endocarditis can be fatal. Even with the current, several month long doxycycline and hydroxychloroquine treatment regimen, the mortality rate is 19%. Further, the drugs have several contraindications, and prolonged use poses significant side effects. Hence, designing better treatment alternatives is crucial. To this end, it is important to understand the pathogenesis of Coxiella endocarditis. The occurrence of endocarditis several years after initial infection suggests that Coxiella successfully subverts the host immune response. Our overall goal is to elucidate the specific host immune pathways Coxiella modulates to facilitate long-term intracellular survival and identify potential therapeutic targets. Previous studies in endocarditis patients demonstrated that increased lipid immune mediator prostaglandin E2 (PGE2) levels contribute to immunosuppression and promote Coxiella growth. Our preliminary studies show increased PGE2 production in Coxiella-infected mouse alveolar macrophages suggesting PGE2 importance in Coxiella pathogenesis and intracellular survival. Since PGE2 production directly correlates to the enzyme cyclooxygenase (Cox) expression and activity, we measured the expression of constitutive Cox-1 and inducible Cox-2 genes. Compared to uninfected cells, Cox-2 but not Cox-1 was upregulated in Coxiella-infected alveolar macrophages. Hence, we hypothesize that inhibition of Cox-2 activity blocks Coxiella intracellular growth. To test this we are currently treating Coxiella-infected cells with FDA-approved Cox-2 inhibitor celecoxib and pan-Cox inhibitor aspirin. Compared to untreated cells, our preliminary qualitative microscopic analysis suggests Coxiella growth inhibition in aspirin-treated cells. Ongoing studies are quantifying changes in Coxiella growth with celecoxib treatment. Future studies will elucidate the mechanisms Coxiella employs to regulate Cox-2 expression and PGE2 production to induce immunosuppression

Role of lipid droplets and prostaglandinE2 in Coxiella burnetii intracellular growth

The obligate intracellular bacterium Coxiella burnetii causes potentially fatal endocarditis several years after initial infection suggesting the bacterium’s ability to persist long-term in the host. Our overall goal is to determine the mechanisms Coxiella employs for its long-term intracellular survival. While the bacterium initially infects alveolar macrophages, in endocarditis patients Coxiella is found in foamy macrophages rich in neutral lipid storage organelles called lipid droplets (LDs). Our previous studies show that Coxiella manipulates host LD metabolism via the Type 4 Secretion System (T4SS), a major virulence factor which secretes bacterial effector proteins into the host cell cytoplasm to manipulate cellular processes. Additionally, blocking LD breakdown almost completely inhibits bacterial growth suggesting that LD-derived lipids are critical for Coxiella intracellular survival. LD breakdown releases arachidonic acids, precursors for the lipid immune mediator prostaglandin E2 (PGE2) which promotes an immunosuppressive environment in alveolar macrophages. We hypothesize that Coxiella manipulates host cell LD metabolism to promote a PGE2-mediated immunosuppressive environment and survive long-term in the host. To test this, we quantified gene expression of PGE2 synthesis enzyme cyclooxygenase-2 (cox-2) in Coxiella-infected alveolar macrophages. Compared to uninfected cells, cox-2 was upregulated in Coxiella-infected but not T4SS mutant-infected macrophages. ELISA showed Coxiella-dependent increase in PGE2 levels indicating that Coxiella T4SS actively manipulates cox-2 expression resulting in increased PGE2 production. Further, blocking PGE2 production using FDA-approved COX-2 inhibitors significantly decreased Coxiella intracellular growth suggesting the importance of PGE2 during Coxiella infection. Ongoing studies are identifying the direct correlation between LDs and PGE2 production and the contribution of LDs to immunosuppression during Coxiella infection. Future studies will determine the potential of blocking PGE2 production as a supplemental therapy for Coxiella endocarditis

Altering lipid droplet homeostasis affects Coxiella burnetii intracellular growth.

Coxiella burnetii is an obligate intracellular bacterial pathogen and a causative agent of culture-negative endocarditis. While C. burnetii initially infects alveolar macrophages, it has also been found in lipid droplet (LD)-containing foamy macrophages in the cardiac valves of endocarditis patients. In addition, transcriptional studies of C. burnetii-infected macrophages reported differential regulation of the LD coat protein-encoding gene perilipin 2 (plin-2). To further investigate the relationship between LDs and C. burnetii, we compared LD numbers using fluorescence microscopy in mock-infected and C. burnetii-infected alveolar macrophages. On average, C. burnetii-infected macrophages contained twice as many LDs as mock-infected macrophages. LD numbers increased as early as 24 hours post-infection, an effect reversed by blocking C. burnetii protein synthesis. The observed LD accumulation was dependent on the C. burnetii Type 4B Secretion System (T4BSS), a major virulence factor that manipulates host cellular processes by secreting bacterial effector proteins into the host cell cytoplasm. To determine the importance of LDs during C. burnetii infection, we manipulated LD homeostasis and assessed C. burnetii intracellular growth. Surprisingly, blocking LD formation with the pharmacological inhibitors triacsin C or T863, or knocking out acyl-CoA transferase-1 (acat-1) in alveolar macrophages, increased C. burnetii growth at least 2-fold. Conversely, preventing LD lipolysis by inhibiting adipose triglyceride lipase (ATGL) with atglistatin almost completely blocked bacterial growth, suggesting LD breakdown is essential for C. burnetii. Together these data suggest that maintenance of LD homeostasis, possibly via the C. burnetii T4BSS, is critical for bacterial growth