Background: Many genes and enzymes of lipid metabolism in T. gondii remain uncharacterized. Resul... more Background: Many genes and enzymes of lipid metabolism in T. gondii remain uncharacterized. Results: The parasite secretes a soluble phosphatidylserine decarboxylase (TgPSD1), which acts on biological membranes. Conclusion: T. gondii uses its secretory apparatus to modify lipids in the parasitophorous vacuole membrane and host cell membranes. Significance: Secreted TgPSD1 reduces externalized phosphatidylserine on host cells, enabling evasion of phagocytosis. Toxoplasma gondii is an obligate intracellular parasite capable of causing fatal infections in immunocompromised individuals and neonates. Examination of the phosphatidylserine (Ptd-Ser) metabolism of T. gondii reveals that the parasite secretes a soluble form of PtdSer decarboxylase (TgPSD1), which preferentially decarboxylates liposomal PtdSer with an apparent K m of 67 M. The specific enzyme activity increases by 3-fold during the replication of T. gondii, and soluble phosphatidylserine decarboxylase (PSD) accounts for ϳ20% of the total PSD, prior to the parasite egress from the host cells. Extracellular T. gondii secreted ϳ20% of its total PSD activity at 37°C, and the intracellular Ca 2؉ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid tetrakis (acetoxymethyl ester) inhibited the process by 50%. Cycloheximide, brefeldin A, ionic composition of the medium, and exogenous PtdSer did not modulate the enzyme secretion, which suggests a constitutive discharge of a presynthesized pool of PSD in axenic T. gondii. TgPSD1 consists of 968 amino acids with a 26-amino acid hydrophobic peptide at the N terminus and no predicted membrane domains. Parasites overexpressing TgPSD1-HA secreted 10-fold more activity compared with the parental strain. Exposure of apoptotic Jurkat cells to transgenic parasites demonstrated interfacial catalysis by secreted TgPSD1 that reduced host cell surface exposure of PtdSer. Immunolocalization experiments revealed that TgPSD1 resides in the dense granules of T. gondii and is also found in the parasitophorous vacuole of replicating parasites. Together, these findings demonstrate novel features of the parasite enzyme because a secreted, soluble, and interfacially active form of PSD has not been previously described for any organism.
The first step in platelet-activating factor production and arachidonic acid release from stimula... more The first step in platelet-activating factor production and arachidonic acid release from stimulated inflammatory cells is thought to be the activation of a phospholipase Aâ (PLAâ). The mouse macrophage cell line, RAW 264, was found to contain PLAâ activity that hydrolyzed ³H-arachidonic acid from sonicated dispersions of 1-0-alkyl-linked phosphatidylcholine (PC). The PLAâ was cytosolic, required calcium, and exhibited a broad
Glutathione (GSH) transport is vital for maintenance of intracellular and extracellular redox bal... more Glutathione (GSH) transport is vital for maintenance of intracellular and extracellular redox balance. Only a few human proteins have been identified as transporters of GSH, glutathione disulfide (GSSG) and/or GSH conjugates (GS-X). Human epithelial MDA1586, A549, H1975, H460, HN4, and H157 cell lines were exposed to 2,5-dihydroxychalcone, which induces a GSH efflux response. A real-time gene superarray for 84 proteins found in families that have a known role in GSH, GSSG, and/or GS-X transport was employed to help identify potential GSH transporters. ABCG2 was identified as the only gene in the array that closely corresponded with the magnitude of 2,5-dihydroxychalcone (2,5-DHC)-induced GSH efflux. The role of human ABCG2 as a novel GSH transporter was verified in a Saccharomyces cerevisiae galactose-inducible gene expression system. Yeast expressing human ABCG2 had 2.5-fold more extracellular GSH compared with those not expressing ABCG2. GSH efflux in ABCG2-expressing yeast was abolished by the ABCG2 substrate methotrexate (10 M), indicating competitive inhibition. In contrast, 2,5-DHC treatment of ABCG2-expressing yeast increased extracellular GSH levels in a dose-dependent manner with a maximum 3.5-fold increase in GSH after 24 h. In addition, suppression of ABCG2 with short hairpin RNA or ABCG2 overexpression in human epithelial cells decreased or increased extracellular GSH levels, respectively. Our data indicate that ABCG2 is a novel GSH transporter.
The negative immune regulator Tollip inhibits the proinflammatory response to rhinovirus (RV) inf... more The negative immune regulator Tollip inhibits the proinflammatory response to rhinovirus (RV) infection, a contributor to airway neutrophilic inflammation and asthma exacerbations, but the underlying molecular mechanisms are poorly understood. Tollip may inhibit IRAK1, a signaling molecule downstream of ST2, the receptor of IL-33. This study was carried out to determine whether Tollip downregulates ST2 signaling via inhibition of IRAK1, but promotes soluble ST2 (sST2) production, thereby limiting excessive IL-8 production in human airway epithelial cells during RV infection in a type 2 cytokine milieu (e.g., IL-13 and IL-33 stimulation). Tollip- and IRAK1-deficient primary human tracheobronchial epithelial (HTBE) cells and Tollip knockout (KO) HTBE cells were generated using the shRNA knockdown and CRISPR/Cas9 approaches, respectively. Cells were stimulated with IL-13, IL-33, and/or RV16. sST2, activated IRAK1, and IL-8 were measured. A Tollip KO mouse model was utilized to test if ...
The exaggerated inflammatory response of asthmatic epithelial cells to in-vitro challenge with My... more The exaggerated inflammatory response of asthmatic epithelial cells to in-vitro challenge with Mycoplasma pneumoniae (MP) has been previously described. Surfactant protein A (SP-A) attenuates this inflammatory response in nonasthmatic epithelial cells. Hypothesis: SP-A derived from patients with asthma is less effective in modulating inflammation than SP-A from normal subjects. Methods: Airway epithelial cells from 10 subjects with asthma (FEV1: 81 6 5% predicted) and 5 normal subjects (FEV1: 95 6 4% predicted) were cultured. SP-A was isolated from bronchoalveolar lavage by ultracentrifugation. After 14 days at an air–liquid interface, cells were exposed to normal (NSP-A) and asthmatic SP-A (A-SPA) separately or both combined 30 minutes before exposure to MP and incubated for 48 hours. IL-8 was determined by ELISA and MUC5AC mRNA by RT-PCR. Results: MP alone significantly increased MUC5AC and IL-8 expression above negative control only in asthmatic cells (P , 0.05). NSP-A significantly decreased IL-8 and MUC5AC from both groups (P , 0.05), but ASP-A did not attenuate MUC5AC and IL-8 expression significantly in either group compared with MP alone. The combination of NSP-A and ASP-A decreased expression of both mediators to the level of uninfected control, ruling out the presence of an inhibitor (Figure). Conclusions: A-SPA failed to abrogate inflammation associated with infection, suggesting one aspect of impaired innate immunity in asthma.
Proceedings of the National Academy of Sciences, 2013
Significance Malaria, caused by intraerythrocytic protozoan parasites of the genus Plasmodium , i... more Significance Malaria, caused by intraerythrocytic protozoan parasites of the genus Plasmodium , is by far the deadliest and most prevalent parasitic disease. Most fatalities are attributable to infection by Plasmodium falciparum . The transmission of P. falciparum into Anopheles mosquitoes is absolutely dependent on the ability of the parasite to differentiate into mature gametocytes. Here we show that P. falciparum requires the plant-like phosphatidylcholine synthesis machinery, which is fueled by host serine, and its key enzyme, phosphoethanolamine N -methyltransferase (PfPMT), for gametocyte development, maturation, and transmission. We identified several compounds that inhibit PfPMT activity and gametocyte development. These compounds also inhibited parasite asexual replication. These new chemical entities provide scaffolds for future development of dual-function antimalarials that can block both infection and transmission.
Background: Toxoplasma gondii is a common intracellular parasite of diverse host cells. Results: ... more Background: Toxoplasma gondii is a common intracellular parasite of diverse host cells. Results: The parasite can produce phosphatidylethanolamine in its mitochondrion, endoplasmic reticulum, and parasitophorous vacuole, which together allow versatile lipid biogenesis. Conclusion: Multiple routes of lipid synthesis ensure parasite survival in discrete nutrient milieus. Significance: T. gondii offers an instructive model to study membrane biology of parasitic protists. Toxoplasma gondii is a highly prevalent obligate intracellular parasite of the phylum Apicomplexa, which also includes other parasites of clinical and/or veterinary importance, such as Plasmodium, Cryptosporidium, and Eimeria. Acute infection by Toxoplasma is hallmarked by rapid proliferation in its host cells and requires a significant synthesis of parasite membranes. Phosphatidylethanolamine (PtdEtn) is the second major phospholipid class in T. gondii. Here, we reveal that PtdEtn is produced in the parasite mitochondrion and parasitophorous vacuole by decarboxylation of phosphatidylserine (PtdSer) and in the endoplasmic reticulum by fusion of CDP-ethanolamine and diacylglycerol. PtdEtn in the mitochondrion is synthesized by a phosphatidylserine decarboxylase (TgPSD1mt) of the type I class. TgPSD1mt harbors a targeting peptide at its N terminus that is required for the mitochondrial localization but not for the catalytic activity. Ablation of TgPSD1mt expression caused up to 45% growth impairment in the parasite mutant. The PtdEtn content of the mutant was unaffected, however, suggesting the presence of compensatory mechanisms. Indeed, metabolic labeling revealed an increased usage of ethanolamine for PtdEtn synthesis by the mutant. Likewise, depletion of nutrients exacerbated the growth defect (ϳ56%), which was partially restored by ethanolamine. Besides, the survival and residual growth of the TgPSD1mt mutant in the nutrient-depleted medium also indicated additional routes of PtdEtn biogenesis, such as acquisition of host-derived lipid. Collectively, the work demonstrates a metabolic cooperativity between the parasite organelles, which ensures a sustained lipid synthesis, survival and growth of T. gondii in varying nutritional milieus.
Background Pulmonary surfactant D (SP-D) has important regulatory functions for innate immunity a... more Background Pulmonary surfactant D (SP-D) has important regulatory functions for innate immunity and has been implicated as a biomarker for chronic obstructive pulmonary disease (COPD). We hypothesized that COPD patients would have reduced bronchoalveolar lavage (BAL) fluid SP-D levels compared to healthy smoking and non-smoking controls. Methods BAL SP-D and phospholipids were quantified and corrected for dilution in 110 subjects (65 healthy never smokers, 23 smokers with normal spirometry, and 22 smokers with COPD). Results BAL SP-D was highest in never smokers (mean 51.9 μg/mL ± 7.1 μg/mL standard error) compared to both smokers with normal spirometry (16.0 μg/mL ± 11.8 μg/mL) and subjects with COPD (19.1 μg/mL ± 12.9 μg/mL; P < 0.0001). Among smokers with COPD, BAL SP-D correlated significantly with FEV1% predicted (R = 0.43; P < 0.05); however, the strongest predictor of BAL SP-D was smoking status. BAL SP-D levels were lowest in current smokers (12.8 μg/mL ± 11.0 μg/mL), ...
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1988
We used the pH-sensitive fluorescent probe 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF... more We used the pH-sensitive fluorescent probe 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) to identify Na+/H + exchange in freshly isolated rat alveolar type II cells and alveolar type II cells in primary culture. The intracellular pH (pHi) of freshly isolated alveolar type II cells was 7.36 -t-0.05 (n = 3). When freshly isolated alveolar type II cells were acid loaded with nigericin in sodium-free buffer, the pH i dropped to 6.59 + 0.04 and remained low in sodium-free buffer. When acid-loaded cells were subsequently incubated with NaCI, pH i increased in a dose-dependent manner. Amiloride (0.1 mM) inhibited the sodium-induced increase in pH i-When the acid-loaded cells were resuspended in an unbuffered choline chloride solution, the cells secreted H + in a sodium-dependent and amiloride-inhibitable manner. Alveolar type II cell monolayers, which were cultured for 22 h on glass coverslips and then loaded with BCECF, had a resting pH i of 7.48 + 0.05 (n = 4). Nigericin acidified these cultured cells in the absence of sodium and NaC! increased the pH i of these acid loaded cells as observed in freshly isolated cells. Secretagogues of pulmonary surfactant, 12-O-tetradecanoylphorbol 13-acetate (TPA) and terbutaline, did not change pH i. Inhibition of the Na+/H + antiporter by the addition of amiloride to a Na + containing medium or the substitution of choline for Na + did not inhibit stimulated phosphatidylcholine secretion. We conclude that pH i regulation in rat alveolar type II cells is in part mediated by an amiloride-sensitive Na+/H + antiporter, but this system appears not to be involved in TPA-or terbutaline-induced pulmonary surfactant secretion in primary culture.
Surfactant protein A (SP-A) regulates a variety of immune cell functions. We determined the abili... more Surfactant protein A (SP-A) regulates a variety of immune cell functions. We determined the ability of SP-A derived from normal and asthmatic subjects to modulate the inflammatory response elicited by Mycoplasma pneumoniae , a pathogen known to exacerbate asthma. Fourteen asthmatic and 10 normal control subjects underwent bronchoscopy with airway brushing and bronchoalveolar lavage (BAL). Total SP-A was extracted from BAL. The ratio of SP-A1 to total SP-A (SP-A1/SP-A) and the binding of total SP-A to M. pneumoniae membranes were determined. Airway epithelial cells from subjects were exposed to either normal or asthmatic SP-A before exposure to M. pneumoniae . IL-8 protein and MUC5AC mRNA were measured. Total BAL SP-A concentration did not differ between groups, but the percentage SP-A1 was significantly increased in BAL of asthmatic compared with normal subjects. SP-A1/SP-A significantly correlated with maximum binding of total SP-A to M. pneumoniae , but only in asthma. SP-A derive...
Lung surfactant protein D (SP-D) binds to Mycobacterium tuberculosis surface lipoarabinomannan an... more Lung surfactant protein D (SP-D) binds to Mycobacterium tuberculosis surface lipoarabinomannan and results in bacterial agglutination, reduced uptake, and inhibition of growth in human macrophages. Here we show that SP-D limits the intracellular growth of bacilli in macrophages by increasing phagosome-lysosome fusion but not by generating a respiratory burst.
Proceedings of the National Academy of Sciences, 2013
Efficient transmission of Plasmodium species between humans and Anopheles mosquitoes is a major c... more Efficient transmission of Plasmodium species between humans and Anopheles mosquitoes is a major contributor to the global burden of malaria. Gametocytogenesis, the process by which parasites switch from asexual replication within human erythrocytes to produce male and female gametocytes, is a critical step in malaria transmission and Plasmodium genetic diversity. Nothing is known about the pathways that regulate gametocytogenesis and only few of the current drugs that inhibit asexual replication are also capable of inhibiting gametocyte development and blocking malaria transmission. Here we provide genetic and pharmacological evidence indicating that the pathway for synthesis of phosphatidylcholine in Plasmodium falciparum membranes from host serine is essential for parasite gametocytogenesis and malaria transmission. Parasites lacking the phosphoethanolamine N-methyltransferase enzyme, which catalyzes the limiting step in this pathway, are severely altered in gametocyte development, are incapable of producing mature-stage gametocytes, and are not transmitted to mosquitoes. Chemical screening identified 11 inhibitors of phosphoethanolamine N-methyltransferase that block parasite intraerythrocytic asexual replication and gametocyte differentiation in the low micromolar range. Kinetic studies in vitro as well as functional complementation assays and lipid metabolic analyses in vivo on the most promising inhibitor NSC-158011 further demonstrated the specificity of inhibition. These studies set the stage for further optimization of NSC-158011 for development of a class of dual activity antimalarials to block both intraerythrocytic asexual replication and gametocytogenesis.
Background: Many genes and enzymes of lipid metabolism in T. gondii remain uncharacterized. Resul... more Background: Many genes and enzymes of lipid metabolism in T. gondii remain uncharacterized. Results: The parasite secretes a soluble phosphatidylserine decarboxylase (TgPSD1), which acts on biological membranes. Conclusion: T. gondii uses its secretory apparatus to modify lipids in the parasitophorous vacuole membrane and host cell membranes. Significance: Secreted TgPSD1 reduces externalized phosphatidylserine on host cells, enabling evasion of phagocytosis. Toxoplasma gondii is an obligate intracellular parasite capable of causing fatal infections in immunocompromised individuals and neonates. Examination of the phosphatidylserine (Ptd-Ser) metabolism of T. gondii reveals that the parasite secretes a soluble form of PtdSer decarboxylase (TgPSD1), which preferentially decarboxylates liposomal PtdSer with an apparent K m of 67 M. The specific enzyme activity increases by 3-fold during the replication of T. gondii, and soluble phosphatidylserine decarboxylase (PSD) accounts for ϳ20% of the total PSD, prior to the parasite egress from the host cells. Extracellular T. gondii secreted ϳ20% of its total PSD activity at 37°C, and the intracellular Ca 2؉ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid tetrakis (acetoxymethyl ester) inhibited the process by 50%. Cycloheximide, brefeldin A, ionic composition of the medium, and exogenous PtdSer did not modulate the enzyme secretion, which suggests a constitutive discharge of a presynthesized pool of PSD in axenic T. gondii. TgPSD1 consists of 968 amino acids with a 26-amino acid hydrophobic peptide at the N terminus and no predicted membrane domains. Parasites overexpressing TgPSD1-HA secreted 10-fold more activity compared with the parental strain. Exposure of apoptotic Jurkat cells to transgenic parasites demonstrated interfacial catalysis by secreted TgPSD1 that reduced host cell surface exposure of PtdSer. Immunolocalization experiments revealed that TgPSD1 resides in the dense granules of T. gondii and is also found in the parasitophorous vacuole of replicating parasites. Together, these findings demonstrate novel features of the parasite enzyme because a secreted, soluble, and interfacially active form of PSD has not been previously described for any organism.
The first step in platelet-activating factor production and arachidonic acid release from stimula... more The first step in platelet-activating factor production and arachidonic acid release from stimulated inflammatory cells is thought to be the activation of a phospholipase Aâ (PLAâ). The mouse macrophage cell line, RAW 264, was found to contain PLAâ activity that hydrolyzed ³H-arachidonic acid from sonicated dispersions of 1-0-alkyl-linked phosphatidylcholine (PC). The PLAâ was cytosolic, required calcium, and exhibited a broad
Glutathione (GSH) transport is vital for maintenance of intracellular and extracellular redox bal... more Glutathione (GSH) transport is vital for maintenance of intracellular and extracellular redox balance. Only a few human proteins have been identified as transporters of GSH, glutathione disulfide (GSSG) and/or GSH conjugates (GS-X). Human epithelial MDA1586, A549, H1975, H460, HN4, and H157 cell lines were exposed to 2,5-dihydroxychalcone, which induces a GSH efflux response. A real-time gene superarray for 84 proteins found in families that have a known role in GSH, GSSG, and/or GS-X transport was employed to help identify potential GSH transporters. ABCG2 was identified as the only gene in the array that closely corresponded with the magnitude of 2,5-dihydroxychalcone (2,5-DHC)-induced GSH efflux. The role of human ABCG2 as a novel GSH transporter was verified in a Saccharomyces cerevisiae galactose-inducible gene expression system. Yeast expressing human ABCG2 had 2.5-fold more extracellular GSH compared with those not expressing ABCG2. GSH efflux in ABCG2-expressing yeast was abolished by the ABCG2 substrate methotrexate (10 M), indicating competitive inhibition. In contrast, 2,5-DHC treatment of ABCG2-expressing yeast increased extracellular GSH levels in a dose-dependent manner with a maximum 3.5-fold increase in GSH after 24 h. In addition, suppression of ABCG2 with short hairpin RNA or ABCG2 overexpression in human epithelial cells decreased or increased extracellular GSH levels, respectively. Our data indicate that ABCG2 is a novel GSH transporter.
The negative immune regulator Tollip inhibits the proinflammatory response to rhinovirus (RV) inf... more The negative immune regulator Tollip inhibits the proinflammatory response to rhinovirus (RV) infection, a contributor to airway neutrophilic inflammation and asthma exacerbations, but the underlying molecular mechanisms are poorly understood. Tollip may inhibit IRAK1, a signaling molecule downstream of ST2, the receptor of IL-33. This study was carried out to determine whether Tollip downregulates ST2 signaling via inhibition of IRAK1, but promotes soluble ST2 (sST2) production, thereby limiting excessive IL-8 production in human airway epithelial cells during RV infection in a type 2 cytokine milieu (e.g., IL-13 and IL-33 stimulation). Tollip- and IRAK1-deficient primary human tracheobronchial epithelial (HTBE) cells and Tollip knockout (KO) HTBE cells were generated using the shRNA knockdown and CRISPR/Cas9 approaches, respectively. Cells were stimulated with IL-13, IL-33, and/or RV16. sST2, activated IRAK1, and IL-8 were measured. A Tollip KO mouse model was utilized to test if ...
The exaggerated inflammatory response of asthmatic epithelial cells to in-vitro challenge with My... more The exaggerated inflammatory response of asthmatic epithelial cells to in-vitro challenge with Mycoplasma pneumoniae (MP) has been previously described. Surfactant protein A (SP-A) attenuates this inflammatory response in nonasthmatic epithelial cells. Hypothesis: SP-A derived from patients with asthma is less effective in modulating inflammation than SP-A from normal subjects. Methods: Airway epithelial cells from 10 subjects with asthma (FEV1: 81 6 5% predicted) and 5 normal subjects (FEV1: 95 6 4% predicted) were cultured. SP-A was isolated from bronchoalveolar lavage by ultracentrifugation. After 14 days at an air–liquid interface, cells were exposed to normal (NSP-A) and asthmatic SP-A (A-SPA) separately or both combined 30 minutes before exposure to MP and incubated for 48 hours. IL-8 was determined by ELISA and MUC5AC mRNA by RT-PCR. Results: MP alone significantly increased MUC5AC and IL-8 expression above negative control only in asthmatic cells (P , 0.05). NSP-A significantly decreased IL-8 and MUC5AC from both groups (P , 0.05), but ASP-A did not attenuate MUC5AC and IL-8 expression significantly in either group compared with MP alone. The combination of NSP-A and ASP-A decreased expression of both mediators to the level of uninfected control, ruling out the presence of an inhibitor (Figure). Conclusions: A-SPA failed to abrogate inflammation associated with infection, suggesting one aspect of impaired innate immunity in asthma.
Proceedings of the National Academy of Sciences, 2013
Significance Malaria, caused by intraerythrocytic protozoan parasites of the genus Plasmodium , i... more Significance Malaria, caused by intraerythrocytic protozoan parasites of the genus Plasmodium , is by far the deadliest and most prevalent parasitic disease. Most fatalities are attributable to infection by Plasmodium falciparum . The transmission of P. falciparum into Anopheles mosquitoes is absolutely dependent on the ability of the parasite to differentiate into mature gametocytes. Here we show that P. falciparum requires the plant-like phosphatidylcholine synthesis machinery, which is fueled by host serine, and its key enzyme, phosphoethanolamine N -methyltransferase (PfPMT), for gametocyte development, maturation, and transmission. We identified several compounds that inhibit PfPMT activity and gametocyte development. These compounds also inhibited parasite asexual replication. These new chemical entities provide scaffolds for future development of dual-function antimalarials that can block both infection and transmission.
Background: Toxoplasma gondii is a common intracellular parasite of diverse host cells. Results: ... more Background: Toxoplasma gondii is a common intracellular parasite of diverse host cells. Results: The parasite can produce phosphatidylethanolamine in its mitochondrion, endoplasmic reticulum, and parasitophorous vacuole, which together allow versatile lipid biogenesis. Conclusion: Multiple routes of lipid synthesis ensure parasite survival in discrete nutrient milieus. Significance: T. gondii offers an instructive model to study membrane biology of parasitic protists. Toxoplasma gondii is a highly prevalent obligate intracellular parasite of the phylum Apicomplexa, which also includes other parasites of clinical and/or veterinary importance, such as Plasmodium, Cryptosporidium, and Eimeria. Acute infection by Toxoplasma is hallmarked by rapid proliferation in its host cells and requires a significant synthesis of parasite membranes. Phosphatidylethanolamine (PtdEtn) is the second major phospholipid class in T. gondii. Here, we reveal that PtdEtn is produced in the parasite mitochondrion and parasitophorous vacuole by decarboxylation of phosphatidylserine (PtdSer) and in the endoplasmic reticulum by fusion of CDP-ethanolamine and diacylglycerol. PtdEtn in the mitochondrion is synthesized by a phosphatidylserine decarboxylase (TgPSD1mt) of the type I class. TgPSD1mt harbors a targeting peptide at its N terminus that is required for the mitochondrial localization but not for the catalytic activity. Ablation of TgPSD1mt expression caused up to 45% growth impairment in the parasite mutant. The PtdEtn content of the mutant was unaffected, however, suggesting the presence of compensatory mechanisms. Indeed, metabolic labeling revealed an increased usage of ethanolamine for PtdEtn synthesis by the mutant. Likewise, depletion of nutrients exacerbated the growth defect (ϳ56%), which was partially restored by ethanolamine. Besides, the survival and residual growth of the TgPSD1mt mutant in the nutrient-depleted medium also indicated additional routes of PtdEtn biogenesis, such as acquisition of host-derived lipid. Collectively, the work demonstrates a metabolic cooperativity between the parasite organelles, which ensures a sustained lipid synthesis, survival and growth of T. gondii in varying nutritional milieus.
Background Pulmonary surfactant D (SP-D) has important regulatory functions for innate immunity a... more Background Pulmonary surfactant D (SP-D) has important regulatory functions for innate immunity and has been implicated as a biomarker for chronic obstructive pulmonary disease (COPD). We hypothesized that COPD patients would have reduced bronchoalveolar lavage (BAL) fluid SP-D levels compared to healthy smoking and non-smoking controls. Methods BAL SP-D and phospholipids were quantified and corrected for dilution in 110 subjects (65 healthy never smokers, 23 smokers with normal spirometry, and 22 smokers with COPD). Results BAL SP-D was highest in never smokers (mean 51.9 μg/mL ± 7.1 μg/mL standard error) compared to both smokers with normal spirometry (16.0 μg/mL ± 11.8 μg/mL) and subjects with COPD (19.1 μg/mL ± 12.9 μg/mL; P < 0.0001). Among smokers with COPD, BAL SP-D correlated significantly with FEV1% predicted (R = 0.43; P < 0.05); however, the strongest predictor of BAL SP-D was smoking status. BAL SP-D levels were lowest in current smokers (12.8 μg/mL ± 11.0 μg/mL), ...
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1988
We used the pH-sensitive fluorescent probe 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF... more We used the pH-sensitive fluorescent probe 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) to identify Na+/H + exchange in freshly isolated rat alveolar type II cells and alveolar type II cells in primary culture. The intracellular pH (pHi) of freshly isolated alveolar type II cells was 7.36 -t-0.05 (n = 3). When freshly isolated alveolar type II cells were acid loaded with nigericin in sodium-free buffer, the pH i dropped to 6.59 + 0.04 and remained low in sodium-free buffer. When acid-loaded cells were subsequently incubated with NaCI, pH i increased in a dose-dependent manner. Amiloride (0.1 mM) inhibited the sodium-induced increase in pH i-When the acid-loaded cells were resuspended in an unbuffered choline chloride solution, the cells secreted H + in a sodium-dependent and amiloride-inhibitable manner. Alveolar type II cell monolayers, which were cultured for 22 h on glass coverslips and then loaded with BCECF, had a resting pH i of 7.48 + 0.05 (n = 4). Nigericin acidified these cultured cells in the absence of sodium and NaC! increased the pH i of these acid loaded cells as observed in freshly isolated cells. Secretagogues of pulmonary surfactant, 12-O-tetradecanoylphorbol 13-acetate (TPA) and terbutaline, did not change pH i. Inhibition of the Na+/H + antiporter by the addition of amiloride to a Na + containing medium or the substitution of choline for Na + did not inhibit stimulated phosphatidylcholine secretion. We conclude that pH i regulation in rat alveolar type II cells is in part mediated by an amiloride-sensitive Na+/H + antiporter, but this system appears not to be involved in TPA-or terbutaline-induced pulmonary surfactant secretion in primary culture.
Surfactant protein A (SP-A) regulates a variety of immune cell functions. We determined the abili... more Surfactant protein A (SP-A) regulates a variety of immune cell functions. We determined the ability of SP-A derived from normal and asthmatic subjects to modulate the inflammatory response elicited by Mycoplasma pneumoniae , a pathogen known to exacerbate asthma. Fourteen asthmatic and 10 normal control subjects underwent bronchoscopy with airway brushing and bronchoalveolar lavage (BAL). Total SP-A was extracted from BAL. The ratio of SP-A1 to total SP-A (SP-A1/SP-A) and the binding of total SP-A to M. pneumoniae membranes were determined. Airway epithelial cells from subjects were exposed to either normal or asthmatic SP-A before exposure to M. pneumoniae . IL-8 protein and MUC5AC mRNA were measured. Total BAL SP-A concentration did not differ between groups, but the percentage SP-A1 was significantly increased in BAL of asthmatic compared with normal subjects. SP-A1/SP-A significantly correlated with maximum binding of total SP-A to M. pneumoniae , but only in asthma. SP-A derive...
Lung surfactant protein D (SP-D) binds to Mycobacterium tuberculosis surface lipoarabinomannan an... more Lung surfactant protein D (SP-D) binds to Mycobacterium tuberculosis surface lipoarabinomannan and results in bacterial agglutination, reduced uptake, and inhibition of growth in human macrophages. Here we show that SP-D limits the intracellular growth of bacilli in macrophages by increasing phagosome-lysosome fusion but not by generating a respiratory burst.
Proceedings of the National Academy of Sciences, 2013
Efficient transmission of Plasmodium species between humans and Anopheles mosquitoes is a major c... more Efficient transmission of Plasmodium species between humans and Anopheles mosquitoes is a major contributor to the global burden of malaria. Gametocytogenesis, the process by which parasites switch from asexual replication within human erythrocytes to produce male and female gametocytes, is a critical step in malaria transmission and Plasmodium genetic diversity. Nothing is known about the pathways that regulate gametocytogenesis and only few of the current drugs that inhibit asexual replication are also capable of inhibiting gametocyte development and blocking malaria transmission. Here we provide genetic and pharmacological evidence indicating that the pathway for synthesis of phosphatidylcholine in Plasmodium falciparum membranes from host serine is essential for parasite gametocytogenesis and malaria transmission. Parasites lacking the phosphoethanolamine N-methyltransferase enzyme, which catalyzes the limiting step in this pathway, are severely altered in gametocyte development, are incapable of producing mature-stage gametocytes, and are not transmitted to mosquitoes. Chemical screening identified 11 inhibitors of phosphoethanolamine N-methyltransferase that block parasite intraerythrocytic asexual replication and gametocyte differentiation in the low micromolar range. Kinetic studies in vitro as well as functional complementation assays and lipid metabolic analyses in vivo on the most promising inhibitor NSC-158011 further demonstrated the specificity of inhibition. These studies set the stage for further optimization of NSC-158011 for development of a class of dual activity antimalarials to block both intraerythrocytic asexual replication and gametocytogenesis.
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Papers by D. Voelker