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Browsing by Subject "Coxiella"
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Item A Member of an Ancient Family of Bacterial Amino Acids Transporters Contributes to Chlamydia Nutritional Virulence and Immune Evasion(American Society for Microbiology, 2023) Banerjee, Arkaprabha; Sun, Yuan; Muramatsu, Matthew K.; Toh, Evelyn; Nelson, David E.; Microbiology and Immunology, School of MedicineMany obligate intracellular bacteria, including members of the genus Chlamydia, cannot synthesize a variety of amino acids de novo and acquire these from host cells via largely unknown mechanisms. Previously, we determined that a missense mutation in ctl0225, a conserved Chlamydia open reading frame of unknown function, mediated sensitivity to interferon gamma. Here, we show evidence that CTL0225 is a member of the SnatA family of neutral amino acid transporters that contributes to the import of several amino acids into Chlamydia cells. Further, we show that CTL0225 orthologs from two other distantly related obligate intracellular pathogens (Coxiella burnetii and Buchnera aphidicola) are sufficient to import valine into Escherichia coli. We also show that chlamydia infection and interferon exposure have opposing effects on amino acid metabolism, potentially explaining the relationship between CTL0225 and interferon sensitivity. Overall, we show that phylogenetically diverse intracellular pathogens use an ancient family of amino acid transporters to acquire host amino acids and provide another example of how nutritional virulence and immune evasion can be linked in obligate intracellular pathogens.Item Coxiella burnetii Sterol-Modifying Protein Stmp1 Regulates Cholesterol in the Intracellular Niche(American Society for Microbiology, 2022) Clemente, Tatiana M.; Ratnayake, Rochelle; Samanta, Dhritiman; Augusto, Leonardo; Beare, Paul A.; Heinzen, Robert A.; Gilk, Stacey D.; Microbiology and Immunology, School of MedicineCoxiella 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.Item Host Lipid Transport Protein ORP1 Is Necessary for Coxiella burnetii Growth and Vacuole Expansion in Macrophages(American Society for Microbiology, 2023) Schuler, Baleigh; Sladek, Margaret; Gilk, Stacey D.; Microbiology and Immunology, School of MedicineCoxiella 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.Item Manipulation of Host Cholesterol by Obligate Intracellular Bacteria(Frontiers, 2017-05-05) Samanta, Dhritiman; Mulye, Minal; Clemente, Tatiana M.; Justis, Anna V.; Gilk, Stacey D.; Microbiology and Immunology, School of MedicineCholesterol 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.Item The Role of Mammalian Lipid Transport Protein ORP1 During Coxiella Burnetii Infection(2022-05) Schuler, Baleigh Elizabeth; Gilk, Stacey D.; Arrizabalaga, Gustavo; Spinola, Stanley; Harrington, Maureen; Day, RichardCoxiella burnetii is an intracellular bacterium that causes the human disease Q fever. C. burnetii is transmitted from infected animals to humans through inhalation of infectious droplets. Acute Q fever is a flu-like illness lasting 10-14 days. Patients often have respiratory symptoms and present with pneumonia. Patients with suppressed immune systems or valvular heart disease can develop chronic Q fever, which causes endocarditis and vasculitis long after initial infection. Chronic Q fever is difficult to treat, and if untreated, is typically fatal. Currently, the United States lacks any vaccine for Q fever. In order to better prevent and treat this disease, it is important to understand how C. burnetii interacts with mammalian cells. Within the host cell, C. burnetii forms a large, acidic Coxiella-containing vacuole (CCV) and uses a Type 4B secretion system (T4SS) 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 is critical for infection. The mammalian lipid transport protein ORP1L localizes to the CCV membrane and mediates CCV-ER membrane contact sites. ORP1L functions in lipid transport, including cholesterol efflux from late endosomes/lysosomes. Its sister isoform ORP1S binds cholesterol but localizes to the cytoplasm and nucleus. In ORP1- null cells, we found that CCVs were smaller than in wildtype cells, highlighting the importance of ORP1 in CCV development. CCVs in ORP1-null cells had higher cholesterol content than CCVs in wildtype cells, suggesting ORP1 functions in cholesterol efflux from the CCV. ORP1-null MH-S cells do not accumulate lipid droplets upon C. burnetii infection, supporting our hypothesis that ORP1 promotes cholesterol transfer from the CCV to the ER, as lipid droplets form from neutral lipids in the ER. 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 demonstrate 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.