Establishing the intracellular niche of obligate intracellular vacuolar pathogens.

Obligate intracellular pathogens occupy one of two niches - free in the host cell cytoplasm or confined in a membrane-bound vacuole. Pathogens occupying membrane-bound vacuoles are sequestered from the innate immune system and have an extra layer of protection from antimicrobial drugs. However, this lifestyle presents several challenges. First, the bacteria must obtain membrane or membrane components to support vacuole expansion and provide space for the increasing bacteria numbers during the log phase of replication. Second, the vacuole microenvironment must be suitable for the unique metabolic needs of the pathogen. Third, as most obligate intracellular bacterial pathogens have undergone genomic reduction and are not capable of full metabolic independence, the bacteria must have mechanisms to obtain essential nutrients and resources from the host cell. Finally, because they are separated from the host cell by the vacuole membrane, the bacteria must possess mechanisms to manipulate the host cell, typically through a specialized secretion system which crosses the vacuole membrane. While there are common themes, each bacterial pathogen utilizes unique approach to establishing and maintaining their intracellular niches. In this review, we focus on the vacuole-bound intracellular niches of Anaplasma phagocytophilum, Ehrlichia chaffeensis, Chlamydia trachomatis, and Coxiella burnetii.

Host Lipid Transport Protein ORP1 Is Necessary for Coxiella burnetii Growth and Vacuole Expansion in Macrophages.

Coxiella burnetii is an intracellular bacterium that causes the human disease Q fever. C. burnetii forms a large, acidic Coxiella-containing vacuole (CCV) and uses a type 4B secretion system to secrete effector proteins into the host cell cytoplasm. While the CCV membrane is rich in sterols, cholesterol accumulation in the CCV is bacteriolytic, suggesting that C. burnetii regulation of lipid transport and metabolism is critical for successful infection. The mammalian lipid transport protein ORP1L (oxysterol binding protein-like protein 1 Long) localizes to the CCV membrane and mediates CCV-endoplasmic reticulum (ER) membrane contact sites. ORP1L functions in lipid sensing and transport, including cholesterol efflux from late endosomes and lysosomes (LELs), and the ER. Its sister isoform, ORP1S (oxysterol binding protein-like protein 1 Short) also binds cholesterol but has cytoplasmic and nuclear localization. In ORP1-null cells, we found that CCVs were smaller than in wild-type cells, highlighting the importance of ORP1 in CCV development. This effect was consistent between HeLa cells and murine alveolar macrophages (MH-S cells). CCVs in ORP1-null cells had higher cholesterol content than CCVs in wild-type cells at 4 days of infection, suggesting ORP1 functions in cholesterol efflux from the CCV. While the absence of ORP1 led to a C. burnetii growth defect in MH-S cells, there was no growth defect in HeLa cells. Together, our data demonstrated that C. burnetii uses the host sterol transport protein ORP1 to promote CCV development, potentially by using ORP1 to facilitate cholesterol efflux from the CCV to diminish the bacteriolytic effects of cholesterol. IMPORTANCE Coxiella burnetii is an emerging zoonotic pathogen and bioterrorism threat. No licensed vaccine exists in the United States, and the chronic form of the disease is difficult to treat and potentially lethal. Postinfectious sequelae of C. burnetii infection, including debilitating fatigue, place a significant burden on individuals and communities recovering from an outbreak. C. burnetii must manipulate host cell processes in order to promote infection. Our results establish a link between host cell lipid transport processes and C. burnetii's avoidance of cholesterol toxicity during infection of alveolar macrophages. Elucidating the mechanisms behind bacterial manipulation of the host will yield insight for new strategies to combat this intracellular pathogen.

Coxiella burnetii actively blocks IL-17-induced oxidative stress in macrophages.

Coxiella burnetii is a highly infectious pathogen that causes Q fever, a leading cause of culture-negative endocarditis. Coxiella first targets alveolar macrophages and forms a phagolysosome-like compartment called the Coxiella-Containing Vacuole (CCV). Successful host cell infection requires the Type 4B Secretion System (T4BSS), which translocates bacterial effector proteins across the CCV membrane into the host cytoplasm, where they manipulate numerous cell processes. Our prior transcriptional studies revealed that Coxiella T4BSS blocks IL-17 signaling in macrophages. Given that IL-17 is known to protect against pulmonary pathogens, we hypothesize that C. burnetii T4BSS downregulates intracellular IL-17 signaling to evade the host immune response and promote bacterial pathogenesis. Using a stable IL-17 promoter reporter cell line, we confirmed that Coxiella T4BSS blocks IL-17 transcription activation. Assessment of the phosphorylation state of NF-κB, MAPK, and JNK revealed that Coxiella downregulates IL-17 activation of these proteins. Using ACT1 knockdown and IL-17RA or TRAF6 knockout cells, we next determined that IL17RA-ACT1-TRAF6 pathway is essential for the IL-17 bactericidal effect in macrophages. In addition, macrophages stimulated with IL-17 generate higher levels of reactive oxygen species, which is likely connected to the bactericidal effect of IL-17. However, C. burnetii T4SS effector proteins block the IL-17-mediated oxidative stress, suggesting that Coxiella blocks IL-17 signaling to avoid direct killing by the macrophages.

The novel bacterial effector protein CbEPF1 mediates ER-LD membrane contacts to regulate host lipid droplet metabolism.

Effective intracellular communication between cellular organelles is pivotal for maintaining cellular homeostasis. Tether proteins, which are responsible for establishing membrane contact sites between cell organelles, enable direct communication between organelles and ultimately influence organelle function and host cell homeostasis. While recent research has identified tether proteins in several bacterial pathogens, their functions have predominantly been associated with mediating inter-organelle communication specifically between the bacteria containing vacuole (BCV) and the host endoplasmic reticulum (ER). However, this study reveals a novel bacterial effector protein, CbEPF1, which acts as a molecular tether beyond the confines of the BCV and facilitates interactions between host cell organelles. Coxiella burnetii, an obligate intracellular bacterial pathogen, encodes the FFAT motif-containing protein CbEPF1 which localizes to host lipid droplets (LDs). CbEPF1 establishes inter-organelle contact sites between host LDs and the ER through its interactions with VAP family proteins. Intriguingly, CbEPF1 modulates growth of host LDs in a FFAT motif-dependent manner. These findings highlight the potential for bacterial effector proteins to impact host cellular homeostasis by manipulating inter-organelle communication beyond conventional BCVs.

Coxiella burnetii Sterol-Modifying Protein Stmp1 Regulates Cholesterol in the Intracellular Niche.

Coxiella burnetii replicates in a phagolysosome-like vacuole called the Coxiella-containing vacuole (CCV). While host cholesterol readily traffics to the CCV, cholesterol accumulation leads to CCV acidification and bacterial death. Thus, bacterial regulation of CCV cholesterol content is essential for Coxiella pathogenesis. Coxiella expresses a sterol-modifying protein, Stmp1, that may function to lower CCV cholesterol through enzymatic modification. Using an Stmp1 knockout (Δstmp1), we determined that Stmp1 is not essential for axenic growth. Inside host cells, however, Δstmp1 mutant bacteria form smaller CCVs which accumulate cholesterol, preferentially fuse with lysosomes, and become more acidic, correlating with a significant growth defect. However, in cholesterol-free cells, Δstmp1 mutant bacteria grow similarly to wild-type bacteria but are hypersensitive to cholesterol supplementation. To better understand the underlying mechanism behind the Δstmp1 mutant phenotype, we performed sterol profiling. Surprisingly, we found that Δstmp1 mutant-infected macrophages accumulated the potent cholesterol homeostasis regulator 25-hydroxycholesterol (25-HC). We next determined whether dysregulated 25-HC alters Coxiella infection by treating wild-type Coxiella-infected cells with 25-HC. Similar to the Δstmp1 mutant phenotype, 25-HC increased CCV proteolytic activity and inhibited bacterial growth. Collectively, these data indicate that Stmp1 alters host cholesterol metabolism and is essential to establish a mature CCV which supports Coxiella growth. IMPORTANCE Coxiella burnetii is the causative agent of human Q fever, an emerging infectious disease and significant cause of culture-negative endocarditis. Acute infections are often undiagnosed, there are no licensed vaccines in the United States, and chronic Q fever requires a prolonged antibiotic treatment. Therefore, new treatment and preventive options are critically needed. Coxiella is an obligate intracellular bacterium that replicates within a large acidic phagolysosome-like compartment, the Coxiella-containing vacuole (CCV). We previously discovered that cholesterol accumulation in the CCV increases its acidification, leading to bacterial death. Therefore, in order to survive in this harsh environment, Coxiella likely regulates CCV cholesterol levels. Here, we found that Coxiella sterol modifying protein (Stmp1) facilitates bacterial growth by reducing CCV cholesterol and host cell 25-hydroxycholesterol (25-HC) levels, which prevents excessive CCV fusion with host lysosomes and CCV acidification. This study establishes that Stmp1-mediated regulation of host cholesterol homeostasis is essential for Coxiella intracellular survival.

A Treatment to Eliminate SARS-CoV-2 Replication in Human Airway Epithelial Cells Is Safe for Inhalation as an Aerosol in Healthy Human Subjects.

BACKGROUND: Low airway surface pH is associated with many airway diseases, impairs antimicrobial host defense, and worsens airway inflammation. Inhaled Optate is designed to safely raise airway surface pH and is well tolerated in humans. Raising intracellular pH partially prevents activation of SARS-CoV-2 in primary normal human airway epithelial (NHAE) cells, decreasing viral replication by several mechanisms. METHODS: We grew primary NHAE cells from healthy subjects, infected them with SARS-CoV-2 (isolate USA-WA1/2020), and used clinical Optate at concentrations used in humans in vivo to determine whether Optate would prevent viral infection and replication. Cells were pretreated with Optate or placebo prior to infection (multiplicity of infection = 1), and viral replication was determined with plaque assay and nucleocapsid (N) protein levels. Healthy human subjects also inhaled Optate as part of a Phase 2a safety trial. RESULTS: Optate almost completely prevented viral replication at each time point between 24 h and 120 h, relative to placebo, on both plaque assay and N protein expression (P < .001). Mechanistically, Optate inhibited expression of major endosomal trafficking genes and raised NHAE intracellular pH. Optate had no effect on NHAE cell viability at any time point. Inhaled Optate was well tolerated in 10 normal subjects, with no change in lung function, vital signs, or oxygenation. CONCLUSIONS: Inhaled Optate may be well suited for a clinical trial in patients with pulmonary SARS-CoV-2 infection. However, it is vitally important for patient safety that formulations designed for inhalation with regard to pH, isotonicity, and osmolality be used. An inhalational treatment that safely prevents SARS-CoV-2 viral replication could be helpful for treating patients with pulmonary SARS-CoV-2 infection.

Lipid hijacking: a unifying theme in vector-borne diseases.

Vector-borne illnesses comprise a significant portion of human maladies, representing 17% of global infections. Transmission of vector-borne pathogens to mammals primarily occurs by hematophagous arthropods. It is speculated that blood may provide a unique environment that aids in the replication and pathogenesis of these microbes. Lipids and their derivatives are one component enriched in blood and are essential for microbial survival. For instance, the malarial parasite Plasmodium falciparum and the Lyme disease spirochete Borrelia burgdorferi, among others, have been shown to scavenge and manipulate host lipids for structural support, metabolism, replication, immune evasion, and disease severity. In this Review, we will explore the importance of lipid hijacking for the growth and persistence of these microbes in both mammalian hosts and arthropod vectors.

Coxiella burnetii Type 4B Secretion System-dependent manipulation of endolysosomal maturation is required for bacterial growth.

Upon host cell infection, the obligate intracellular bacterium Coxiella burnetii resides and multiplies within the Coxiella-Containing Vacuole (CCV). The nascent CCV progresses through the endosomal maturation pathway into a phagolysosome, acquiring endosomal and lysosomal markers, as well as acidic pH and active proteases and hydrolases. Approximately 24-48 hours post infection, heterotypic fusion between the CCV and host endosomes/lysosomes leads to CCV expansion and bacterial replication in the mature CCV. Initial CCV acidification is required to activate C. burnetii metabolism and the Type 4B Secretion System (T4BSS), which secretes effector proteins required for CCV maturation. However, we found that the mature CCV is less acidic (pH~5.2) than lysosomes (pH~4.8). Further, inducing CCV acidification to pH~4.8 causes C. burnetii lysis, suggesting C. burnetii actively regulates pH of the mature CCV. Because heterotypic fusion with host endosomes/lysosomes may influence CCV pH, we investigated endosomal maturation in cells infected with wildtype (WT) or T4BSS mutant (ΔdotA) C. burnetii. In WT-infected cells, we observed a significant decrease in proteolytically active, LAMP1-positive endolysosomal vesicles, compared to mock or ΔdotA-infected cells. Using a ratiometric assay to measure endosomal pH, we determined that the average pH of terminal endosomes in WT-infected cells was pH~5.8, compared to pH~4.75 in mock and ΔdotA-infected cells. While endosomes progressively acidified from the periphery (pH~5.5) to the perinuclear area (pH~4.7) in both mock and ΔdotA-infected cells, endosomes did not acidify beyond pH~5.2 in WT-infected cells. Finally, increasing lysosomal biogenesis by overexpressing the transcription factor EB resulted in smaller, more proteolytically active CCVs and a significant decrease in C. burnetii growth, indicating host lysosomes are detrimental to C. burnetii. Overall, our data suggest that C. burnetii inhibits endosomal maturation to reduce the number of proteolytically active lysosomes available for heterotypic fusion with the CCV, possibly as a mechanism to regulate CCV pH.

Functional inhibition of acid sphingomyelinase disrupts infection by intracellular bacterial pathogens.

Intracellular bacteria that live in host cell-derived vacuoles are significant causes of human disease. Parasitism of low-density lipoprotein (LDL) cholesterol is essential for many vacuole-adapted bacteria. Acid sphingomyelinase (ASM) influences LDL cholesterol egress from the lysosome. Using functional inhibitors of ASM (FIASMAs), we show that ASM activity is key for infection cycles of vacuole-adapted bacteria that target cholesterol trafficking-Anaplasma phagocytophilum, Coxiella burnetii, Chlamydia trachomatis, and Chlamydia pneumoniae. Vacuole maturation, replication, and infectious progeny generation by A. phagocytophilum, which exclusively hijacks LDL cholesterol, are halted and C. burnetii, for which lysosomal cholesterol accumulation is bactericidal, is killed by FIASMAs. Infection cycles of Chlamydiae, which hijack LDL cholesterol and other lipid sources, are suppressed but less so than A. phagocytophilum or C. burnetii A. phagocytophilum fails to productively infect ASM-/- or FIASMA-treated mice. These findings establish the importance of ASM for infection by intracellular bacteria and identify FIASMAs as potential host-directed therapies for diseases caused by pathogens that manipulate LDL cholesterol.

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.

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.

Measuring pH of the Coxiella burnetii Parasitophorous Vacuole.

Coxiella burnetii is the causative agent of human Q fever, a zoonotic disease that can cause a debilitating, flu-like illness in acute cases, or a life-threatening endocarditis in chronic patients. An obligate intracellular bacterial pathogen, Coxiella survives and multiplies in a large lysosome-like vacuole known as the Coxiella parasitophorous vacuole (CPV). A unique characteristic of the CPV is the acidic environment (pH ∼5.0), which is required to activate Coxiella metabolism and the Coxiella type 4 secretion system (T4SS), a major virulence factor required for intracellular survival. Further, inhibiting or depleting vacuolar ATPase, a host cell protein that regulates lysosomal pH, inhibits intracellular Coxiella growth. Together, these data suggest that CPV pH is an important limiting factor for Coxiella growth and virulence. This unit describes a method to determine CPV pH using live cell microscopy of a pH-sensitive fluorophore conjugated to dextran. This technique is useful to measure changes in CPV pH during infection or in response to drug treatment. © 2017 by John Wiley & Sons, Inc.

Quantitative Dextran Trafficking to the Coxiella burnetii Parasitophorous Vacuole.

The gram-negative bacterium Coxiella burnetii causes human Q fever, a disease characterized by a debilitating flu-like illness in acute cases and endocarditis in chronic patients. An obligate intracellular pathogen, Coxiella burnetii survives within a large, lysosome-like vacuole inside the host cell. A unique feature of the Coxiella parasitophorous vacuole (PV) is high levels of fusion with the host endocytic pathway, with PV-endosome fusion critical for Coxiella survival within the host cell. This unit describes quantitating PV-endosome fusion by measuring delivery of the fluid phase endosome marker dextran to the PV using live cell imaging. To study the effect of host cell proteins involved in PV-endosome fusion, details are provided for using siRNA knockdown host cells. This method is a powerful tool for understanding mechanisms underlying Coxiella's ability to manipulate host cell trafficking pathways. © 2017 by John Wiley & Sons, Inc.

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.

Interactions between the Coxiella burnetii parasitophorous vacuole and the endoplasmic reticulum involve the host protein ORP1L.

Coxiella burnetii is a gram-negative intracellular bacterium that forms a large, lysosome-like parasitophorous vacuole (PV) essential for bacterial replication. Host membrane lipids are critical for the formation and maintenance of this intracellular niche, yet the mechanisms by which Coxiella manipulates host cell lipid metabolism, trafficking and signalling are unknown. Oxysterol-binding protein-related protein 1 long (ORP1L) is a mammalian lipid-binding protein that plays a dual role in cholesterol-dependent endocytic trafficking as well as interactions between endosomes and the endoplasmic reticulum (ER). We found that ORP1L localized to the Coxiella PV within 12 h of infection through a process requiring the Coxiella Dot/Icm Type 4B secretion system, which secretes effector proteins into the host cell cytoplasm where they manipulate trafficking and signalling pathways. The ORP1L N-terminal ankyrin repeats were necessary and sufficient for PV localization, indicating that ORP1L binds a PV membrane protein. Strikingly, ORP1L simultaneously co-localized with the PV and ER, and electron microscopy revealed membrane contact sites between the PV and ER membranes. In ORP1L-depleted cells, PVs were significantly smaller than PVs from control cells. These data suggest that ORP1L is specifically recruited by the bacteria to the Coxiella PV, where it influences PV membrane dynamics and interactions with the ER.

Preventing friendly fire in the war on microbes.

Lung injury induced by Pseudomonas aeruginosa infection might be subverted by targeting a chromatin-remodeling protein (Zou et al., this issue).

Molecular requirement for sterols in herpes simplex virus entry and infectivity.

Herpes simplex virus 1 (HSV-1) required cholesterol or desmosterol for virion-induced membrane fusion. HSV successfully entered DHCR24(-/-) cells, which lack a desmosterol-to-cholesterol conversion enzyme, indicating that entry can occur independently of cholesterol. Depletion of desmosterol from these cells resulted in diminished HSV-1 entry, suggesting a general sterol requirement for HSV-1 entry and that desmosterol can operate in virus entry. Cholesterol functioned more effectively than desmosterol, suggesting that the hydrocarbon tail of cholesterol influences viral entry.

Bacterial colonization of host cells in the absence of cholesterol.

Reports implicating important roles for cholesterol and cholesterol-rich lipid rafts in host-pathogen interactions have largely employed sterol sequestering agents and biosynthesis inhibitors. Because the pleiotropic effects of these compounds can complicate experimental interpretation, we developed a new model system to investigate cholesterol requirements in pathogen infection utilizing DHCR24(-/-) mouse embryonic fibroblasts (MEFs). DHCR24(-/-) MEFs lack the Δ24 sterol reductase required for the final enzymatic step in cholesterol biosynthesis, and consequently accumulate desmosterol into cellular membranes. Defective lipid raft function by DHCR24(-/-) MEFs adapted to growth in cholesterol-free medium was confirmed by showing deficient uptake of cholera-toxin B and impaired signaling by epidermal growth factor. Infection in the absence of cholesterol was then investigated for three intracellular bacterial pathogens: Coxiella burnetii, Salmonella enterica serovar Typhimurium, and Chlamydia trachomatis. Invasion by S. Typhimurium and C. trachomatis was unaltered in DHCR24(-/-) MEFs. In contrast, C. burnetii entry was significantly decreased in -cholesterol MEFs, and also in +cholesterol MEFs when lipid raft-associated α(V)ß(3) integrin was blocked, suggesting a role for lipid rafts in C. burnetii uptake. Once internalized, all three pathogens established their respective vacuolar niches and replicated normally. However, the C. burnetii-occupied vacuole within DHCR24(-/-) MEFs lacked the CD63-positive material and multilamellar membranes typical of vacuoles formed in wild type cells, indicating cholesterol functions in trafficking of multivesicular bodies to the pathogen vacuole. These data demonstrate that cholesterol is not essential for invasion and intracellular replication by S. Typhimurium and C. trachomatis, but plays a role in C. burnetii-host cell interactions.

Role of lipids in Coxiella burnetii infection.

Lipids are essential components of both eukaryotic and prokaryotic cells, serving diverse functions including energy metabolism and membrane structure. Intracellular vacuolar pathogens such as Coxiella burnetii require lipids for both normal bacterial functions as well as formation of the acidic, phagolysosomal-like parasitophorous vacuole (PV) surrounding the bacteria. As an intracellular pathogen, C. burnetii can acquire lipid through both de novo bacterial synthesis and subversion of host cell pools. The C. burnetii genome encodes enzymes required for de novo synthesis of fatty acids and phospholipids. The high percentage of branched fatty acids suggests C. burnetii modifies these molecules to generate a bacterial cell envelope that can resist the harsh environment of the PV, such as the acidic pH. In addition to fatty acids and their derivatives, C. burnetii requires isoprenoids, particularly sterols as the PV membrane is cholesterol-rich. With the exception of two eukaryote-like sterol reductases, C. burnetii does not have the capability to generate cholesterol, suggesting sterols are actively diverted from the host cell. While C. burnetii utilizes host cell lipids for membrane biogenesis and possibly energy, bacterial manipulation of host cell lipid signaling pathways may support establishment of the intracellular niche. For example, effectors secreted by the C. burnetii Type IV secretion system may either directly or indirectly modify host cell lipids. Further understanding of the lipid biosynthetic capabilities of C. burnetii, along with C. burnetii's manipulation of host cell lipids, will provide insight into the host-pathogen relationship.