Postdoc_research_Day2010
1
Esterase Activity of the Chlamydia trachomatis CT149 Gene Product Results in Cholesteryl Ester Hydrolysis When Expressed in HeLa Cells Introduction Chlamydia trachomatis is an important human pathogen responsible for ocular or genital infections. C. trachomatis serovars D-K are the most common bacterial sexual transmitted diseases world-wide with more than a million new reported cases in 2008 in the USA alone[1]. C. trachomatis is an obligate intracellular bacterial pathogen with a unique biphasic developmental cycle characterized by an extracellular infectious, but metabolic inert elementary body (EB) and an intracellular metabolic active, but non-infectious reticulate body (RB). In eukaryotic host cells chlamydiae are found in a membrane-surrounded vesicle called the inclusion, which is non-fusogenic with lysosomes[2]. After numerous rounds of RB replication differentiation back to EBs occur, releasing infectious forms to invade additional host cells. Chlamydia, like other intracellular bacteria, are auxotrophic for a variety of essential metabolites. For example, productive chlamydial growth requires host cell lipid transport[3] and transported lipids accumulate at the site of chlamydial growth within the developing inclusion[4]. Cholesterol and the cholesterol biosynthetic pathway intermediates are essential for mammalian cells[5]. However, a role for cholesterol in bacterial pathogens has only recently been recognized by demonstrating the presence of cholesterol in membrane lipids for a small subset of microbes[6], and showing a role for cholesterol catabolism as an energy source for Mycobacteria[7]. Modulation of lipid metabolism within infected cells may have pathological significance in chlamydial disease[8,9]; therefore we investigated mechanisms whereby C. trachomatis acquires cholesterol, and utilizes the essential lipid for intracellular growth. Results - The heme-ligand inhibitor 4-phenyl imidazole inhibited chlamydial growth, which was reversed by cholesterol addition. - The Dehydrocholesterol reductase specific inhibitor BM15766 inhibited chlamydial growth only in the absence of exogenous cholesterol. - Knockdown of cyp51A1 with siRNA had no effect on chlamydial growth suggesting that 4-PI also targets a chlamydial protein. - In silico analysis of a putative hydrolase CT149 identified two lipase/esterase GXSXG motifs, a potential cholesterol recognition/interaction amino acid consensus (CRAC) sequence, and an N-terminal, cleavable signal sequence - Lysates of E. coli overexpressing CT149 resulted in ester hydrolysis in an in vitro carboxylic esterase assay - Expression of CT149 in HeLa cells resulted in a decrease of cytoplasmic cholesteryl esters within 48hrs Jan Peters and Gerald I Byrne Department of Microbiology, Immunology & Biochemistry, University of Tennessee Health Science Center, Memphis, TN 38163 Methods HeLa cells were pre- treated with the heme-ligand inhibitor 4-phenyl imidazole (4-PI) and the 7-dehydrocholesterol reductase inhibitor BM15766. Cells were infected with C. trachomatis D and observed after staining by fluorescence microscopy and evaluated by determining the presence of infectious chlamydiae by harvest and titration. HeLa cells were transfected with siRNA against the host gene cyp51A, which codes for Lanosteol-14a- demethylase and is a target of 4-PI. Available online programs were used with default settings to perform in silico analysis of a target chlamydial putative hydrolase CT149. CT149, and an unrelated gene (CT148) as control, were overexpressed in E. coli and lysates used in a cell-free carboxylic esterase assay using ortho-nitrophenyl acetate. CT148 and CT149 also were over-expressed in HeLa cells and lipid extracts measured for cholesterol and cholesterol esters. Gene knockdown of cyp51A1 has no influence on chlamydial growth In silico analysis of a putative hydrolase CT149 References 1. STD surveillance report 2008, CDC website: http://www.cdc.gov/std/stats08/ 2. Moulder, J.W., Interaction of chlamydiae and host cells in vitro. Microbiol Rev, 1991. 55(1): p. 143-90 3. Carabeo, RA, DJ Mead, and T Hackstadt. 2003. Golgi-dependent transport of cholesterol to the Chlamydia trachomatis inclusion. Proc Natl Acad Sci USA. 100: 6771-6776. 4. Cocchiaro, JL, Y Kumar, ER Fischer, T Hackstadt, and RH Valdivia. 2008. Cytoplasmic lipid droplets are translocated into the lumen of the Chlamydia trachomatis parasitophorous vacuole. Proc Natl Acad Sci USA. 105: 9379- 9384. 5. Fernandez, C, M Martin, D Gomez-Coronado, and MA Lasuncion. 2005. Effects of distal cholesterol biosynthesis inhibitors on cell proliferation and cell cycle progression. J Lipid Res. 46: 920-929. 6. Razin, S. 1975. Cholesterol incorporation into bacterial membranes. J Bacteriol. 124: 570-572. 7. Pandey, AK, and CM Sassetti. 2008. Mycobacterial persistence requires the utilization of host cholesterol. Proc Natl Acad Sci USA. 105: 4376-4380. 8. Jutras, I, L Abrami, and A Dautry-Varsat. 2003. Entry of the lymphogranuloma venereum strain of Chlamydia trachomatis into host cells involves cholesterol-rich membrane domains. Infect Immun. 71: 260-266. 9. Jamison, WP, and T Hackstadt. 2008. Induction of type III secretion by cell-free Chlamydia trachomatis elementary bodies. Microbial pathogen. 45: 435-440. Figure Figure 5 - HeLa cells were transfected with 30nM siRNA against cyp51A1 and incubated at 37 C for 48hrs. Cells were harvested, RNA purified and protein lysates in TEN-T buffer prepared. RT-PCR against cyp51A1 or aldoA. In Western blot analysis 5ug total protein were separated on 10% SDS-PAGE gel and proteins detected with antibodies to Cyp51A1 or Aldolase A . HeLa cells were infected with 0.5 MOI for IFA and 1 MOI for IFU titration, and incubated at 37°C for 24 or 48hrs. Cells were methanol fixed in methanol and stained with FITC-conjugated antibody to C. trachomatis. Representative images (200x magnification fluorescence) are shown. For C. trachomatis IFU cells were harvested 24h or 48h p.i. and HeLa cells fresh infected with dilutions. All samples for titration were done in triplicate. Error bars represent standard deviation. Figure 6 - The protein sequence of CT149 was searched with SignalP for bacterial or eukaryotic signal sequences with default settings. A potential cleavage site (black arrow) was identified between amino acids 18 and 19. The lipase/esterase specific protein motif GXSXG, and the CRAC sequence were identified within the hydrolase domain. B) Protein alignment of CT149 and the homologs in C. trachomatis serovar 434/Bu (CTL0404, Acc. no. YP_001654488.1), C. trachomatis A/HAR-13 (CTA_158, Acc. no. YP_327950.1), C. pneumonia AR39 (CP_0620, Acc. no. AAF38435.1), and C. caviae GPIC (CCA_00614, Acc no. AAP05356.1) in ClustalX2 with default settings. The black arrow marks the cleavage site in all sequences. The GXSXG lipase/esterase and the cholesterol recognition/interaction amino acid consensus (CRAC) sequence are marked with a red and black box respectively. Schematic drawing is not in scale. Recombinant expressed CT149 shows carboxylic esterase activity in vitro Figure 7 - Carboxylic esterase activity was measured using ortho-nitrophenyl acetate as substrate and the production of ortho-nitrophenol after hydrolysis monitored at 420nm at RT. A) Carboxylic esterase activity of lysate from E. coli expressing recombinant CT149 was monitored for 30 min. B) Comparison of carboxylic esterase activity in lysates of E. coli with empty vector (black symbols), expressing the unrelated CT148 (blue symbols) or CT149 (red symbols). Esterase activity was measured using 150 (circle), 200 (triangle), and 250ug (square) total protein. Expression of CT149 in HeLa cells resulted in a decrease of cholesteryl esters Figure 8 - HeLa cells were transfected with empty pIRIS2-DsRed2, pIRIS2-DsRed2-CT148 or pIRIS2-DsRed2-CT149 and incubated at 37°C for 48hrs. Lipids were extracted and the amounts of cholesteryl esters, free cholesterol and total cholesterol determined. The experiment was done in triplicate and free and total cholesterol measured in triplicate. The amount of cholesteryl esters was calculated from the difference of total and free cholesterol. Error bars represent standard deviation and a student t-test to calculate statistical significance. Figure 4 - Growth is restored by addition of exogenous cholesterol. Cells were infected with C. trachomatis in the presence or absence of serum containing extracellular cholesterol without or with the inhibitors 4-PI and BM15766. Cells were incubated for 24hrs and the medium was exchanged with fresh medium. For rescue 25ug/ml cholesterol was added directly to the medium. A) Cells were methanol fixed 24h or 48h p.i. Representative images (200x magnification fluorescence) are shown. B) Cells were harvested 24h or 48h p.i. and HeLa cells fresh infected in dilutions to titrate the C. trachomatis IFU. All titrations were done in triplicate. Error bars represent standard deviation. The DHCR7 specific inhibitor BM15766 inhibits chlamydial growth only in the absence of extracellular cholesterol 4-PI induced growth inhibition can be restored after removing the inhibitor or adding exogenous cholesterol Figure 3 - C. trachomatis infected cells with or without 1mM 4-PI were incubated for 24hrs and the medium exchanged with inhibitor, with inhibitor and 25ug/ml cholesterol, or medium without inhibitor and further incubated for additional 24hrs. A) Cells were fixed at 24h or 48h p.i. Representative images (200x magnification fluorescence) are shown. B) Cells were harvested 24h or 48h p.i. and HeLa cells fresh infected with dilutions to titrate the C. trachomatis IFU. All titrations were done in triplicate. Error bars represent standard deviation. Figure 2 - The figure shows the enzyme targets (name in red) of 4-PI and BM15766 (left side) and their catalyzed reaction (right side). The inhibitor 4-PI targets Lanosterol-14 demethylase, which catalyzes the demethylation of 24-dihydrolanosterol to 4,4-dimethylcholesta-8(9),14-dien-3beta-ol (target methyl group in red). The 7- dehydrocholesterol reductase specific inhibitor BM15766 blocks the reduction of one double bond in dehydrocholesterol to cholesterol (target double bond in red), the very last step in the biosynthesis pathway. The IDO-inhibitor 4-phenyl imidazole inhibits C. trachomatis growth independent of IFN- . Figure 1 - Untreated (upper panel) and 50ng/ml IFN- treated (lower panel) cells in the absence or presence of the 1uM L-1-MT, 100uM and 1mM 4-PI were infected after 24hrs with an MOI of 1. After 24h p.i. cells were fixed in methanol and stained with an FITC-conjugated antibody to C. trachomatis D than counterstained with Evans blue. Representative images (200x magnification fluorescence) are shown. Acknowledgement This work was supported by the National Institutes of Health gra nt AI 19782. For further information: Contact Dr. Gerald Byrne: [email protected] Simplified cholesterol biosynthesis pathway in eukaryotic cells Conclusion These results suggest that CT149 has carboxylic esterase activity and contributes to the hydrolysis of eukaryotic cholesteryl esters during intracellular growth. Our data suggest that CT149 plays a role in cholesterol metabolism by Chlamydia and may be a target for chemotherapeutic development. Future direction Substrate specificity of CT149 will be tested by using different cholesteryl esters as well as other lipids including tri-, di, and monoglycerides We will investigate if the inhibition of chlamydial growth is a result of the interaction of 4- Phenyl imidazole with CT149 or if other enzymes are involved. Monoclonal antibodies against CT149 will be produced and used to observe the location and expression within the host cell during the chlamydial infection to different time points in the developmental cycle. A A A A B B B B
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Transcript of Postdoc_research_Day2010
Esterase Activity of the Chlamydia trachomatis CT149 Gene Product Results inCholesteryl Ester Hydrolysis When Expressed in HeLa Cells
IntroductionChlamydia trachomatis is an important human pathogen responsible for ocular orgenital infections. C. trachomatis serovars D-K are the most common bacterial sexualtransmitted diseases world-wide with more than a million new reported cases in 2008in the USA alone[1].C. trachomatis is an obligate intracellular bacterial pathogen with a unique biphasicdevelopmental cycle characterized by an extracellular infectious, but metabolic inertelementary body (EB) and an intracellular metabolic active, but non-infectious reticulatebody (RB). In eukaryotic host cells chlamydiae are found in a membrane-surroundedvesicle called the inclusion, which is non-fusogenic with lysosomes[2]. After numerousrounds of RB replication differentiation back to EBs occur, releasing infectious formsto invade additional host cells.Chlamydia, like other intracellular bacteria, are auxotrophic for a variety of essentialmetabolites. For example, productive chlamydial growth requires host cell lipidtransport[3] and transported lipids accumulate at the site of chlamydial growth withinthe developing inclusion[4]. Cholesterol and the cholesterol biosynthetic pathwayintermediates are essential for mammalian cells[5]. However, a role for cholesterol inbacterial pathogens has only recently been recognized by demonstrating the presenceof cholesterol in membrane lipids for a small subset of microbes[6], and showing arole for cholesterol catabolism as an energy source for Mycobacteria[7]. Modulationof lipid metabolism within infected cells may have pathological significance in chlamydialdisease[8,9]; therefore we investigated mechanisms whereby C. trachomatis acquirescholesterol, and utilizes the essential lipid for intracellular growth.
Results- The heme-ligand inhibitor 4-phenyl imidazole inhibited chlamydial growth, which
was reversed by cholesterol addition.- The Dehydrocholesterol reductase specific inhibitor BM15766 inhibited chlamydial
growth only in the absence of exogenous cholesterol.- Knockdown of cyp51A1 with siRNA had no effect on chlamydial growth suggesting
that 4-PI also targets a chlamydial protein.- In silico analysis of a putative hydrolase CT149 identified two lipase/esterase GXSXG
motifs, a potential cholesterol recognition/interaction amino acid consensus (CRAC)sequence, and an N-terminal, cleavable signal sequence
- Lysates of E. coli overexpressing CT149 resulted in ester hydrolysis in an in vitro carboxylic esterase assay
- Expression of CT149 in HeLa cells resulted in a decrease of cytoplasmic cholesterylesters within 48hrs
Jan Peters and Gerald I ByrneDepartment of Microbiology, Immunology & Biochemistry, University of Tennessee Health Science Center, Memphis, TN 38163
MethodsHeLa cells were pre- treated with the heme-ligand inhibitor 4-phenyl imidazole (4-PI) and the7-dehydrocholesterol reductase inhibitor BM15766. Cells were infected with C. trachomatisD and observed after staining by �uorescence microscopy and evaluated by determining thepresence of infectious chlamydiae by harvest and titration. HeLa cells were transfected withsiRNA against the host gene cyp51A, which codes for Lanosteol-14a- demethylase and is atarget of 4-PI. Available online programs were used with default settings to perform in silicoanalysis of a target chlamydial putative hydrolase CT149. CT149, and an unrelated gene(CT148) as control, were overexpressed in E. coli and lysates used in a cell-free carboxylicesterase assay using ortho-nitrophenyl acetate. CT148 and CT149 also were over-expressedin HeLa cells and lipid extracts measured for cholesterol and cholesterol esters.
Gene knockdown of cyp51A1 has no influenceon chlamydial growth
In silico analysis of a putative hydrolase CT149
References1. STD surveillance report 2008, CDC website: http://www.cdc.gov/std/stats08/2. Moulder, J.W., Interaction of chlamydiae and host cells in vitro. Microbiol Rev, 1991. 55(1): p. 143-903. Carabeo, RA, DJ Mead, and T Hackstadt. 2003. Golgi-dependent transport of cholesterol to the Chlamydia
trachomatis inclusion. Proc Natl Acad Sci USA. 100: 6771-6776.4. Cocchiaro, JL, Y Kumar, ER Fischer, T Hackstadt, and RH Valdivia. 2008. Cytoplasmic lipid droplets are
translocated into the lumen of the Chlamydia trachomatis parasitophorous vacuole. Proc Natl Acad Sci USA. 105: 9379- 9384.
5. Fernandez, C, M Martin, D Gomez-Coronado, and MA Lasuncion. 2005. Effects of distal cholesterol biosynthesisinhibitors on cell proliferation and cell cycle progression. J Lipid Res. 46: 920-929.
6. Razin, S. 1975. Cholesterol incorporation into bacterial membranes. J Bacteriol. 124: 570-572.7. Pandey, AK, and CM Sassetti. 2008. Mycobacterial persistence requires the utilization of host cholesterol. Proc
Natl Acad Sci USA. 105: 4376-4380.8. Jutras, I, L Abrami, and A Dautry-Varsat. 2003. Entry of the lymphogranuloma venereum strain of
Chlamydia trachomatis into host cells involves cholesterol-rich membrane domains. Infect Immun. 71: 260-266.9. Jamison, WP, and T Hackstadt. 2008. Induction of type III secretion by cell-free Chlamydia trachomatis
elementary bodies. Microbial pathogen. 45: 435-440.
FigureFigure 5 - HeLa cells were transfected with 30nM siRNA against cyp51A1 and incubated at 37 C for 48hrs. Cellswere harvested, RNA purified and protein lysates in TEN-T buffer prepared. RT-PCR against cyp51A1 or aldoA. InWestern blot analysis 5ug total protein were separated on 10% SDS-PAGE gel and proteins detected with antibodiesto Cyp51A1 or Aldolase A . HeLa cells were infected with 0.5 MOI for IFA and 1 MOI for IFU titration, and incubatedat 37°C for 24 or 48hrs. Cells were methanol fixed in methanol and stained with FITC-conjugated antibody to C.trachomatis. Representative images (200x magnification fluorescence) are shown. For C. trachomatis IFU cells wereharvested 24h or 48h p.i. and HeLa cells fresh infected with dilutions. All samples for titration were done in triplicate.Error bars represent standard deviation.
Figure 6 - The protein sequence of CT149 was searched with SignalP for bacterial or eukaryotic signal sequenceswith default settings. A potential cleavage site (black arrow) was identified between amino acids 18 and 19. Thelipase/esterase specific protein motif GXSXG, and the CRAC sequence were identified within the hydrolase domain. B) Protein alignment of CT149 and the homologs in C. trachomatis serovar 434/Bu (CTL0404, Acc. no. YP_001654488.1),C. trachomatis A/HAR-13 (CTA_158, Acc. no. YP_327950.1), C. pneumonia AR39 (CP_0620, Acc. no. AAF38435.1),and C. caviae GPIC (CCA_00614, Acc no. AAP05356.1) in ClustalX2 with default settings. The black arrow marksthe cleavage site in all sequences. The GXSXG lipase/esterase and the cholesterol recognition/interaction aminoacid consensus (CRAC) sequence are marked with a red and black box respectively. Schematic drawing is not inscale.
Recombinant expressed CT149 showscarboxylic esterase activity in vitro
Figure 7 - Carboxylic esterase activity was measured using ortho-nitrophenyl acetate as substrate and the productionof ortho-nitrophenol after hydrolysis monitored at 420nm at RT. A) Carboxylic esterase activity of lysate from E.coli expressing recombinant CT149 was monitored for 30 min. B) Comparison of carboxylic esterase activity inlysates of E. coli with empty vector (black symbols), expressing the unrelated CT148 (blue symbols) or CT149 (redsymbols). Esterase activity was measured using 150 (circle), 200 (triangle), and 250ug (square) total protein.
Expression of CT149 in HeLa cells resulted in a decreaseof cholesteryl esters
Figure 8 - HeLa cells were transfected with empty pIRIS2-DsRed2, pIRIS2-DsRed2-CT148 or pIRIS2-DsRed2-CT149and incubated at 37°C for 48hrs. Lipids were extracted and the amounts of cholesteryl esters, free cholesterol andtotal cholesterol determined. The experiment was done in triplicate and free and total cholesterol measured intriplicate. The amount of cholesteryl esters was calculated from the difference of total and free cholesterol. Errorbars represent standard deviation and a student t-test to calculate statistical significance.
Figure 4 - Growth is restored by addition of exogenous cholesterol. Cells were infected with C. trachomatis in thepresence or absence of serum containing extracellular cholesterol without or with the inhibitors 4-PI and BM15766.Cells were incubated for 24hrs and the medium was exchanged with fresh medium. For rescue 25ug/ml cholesterolwas added directly to the medium. A) Cells were methanol fixed 24h or 48h p.i. Representative images (200xmagnification fluorescence) are shown. B) Cells were harvested 24h or 48h p.i. and HeLa cells fresh infected indilutions to titrate the C. trachomatis IFU. All titrations were done in triplicate. Error bars represent standarddeviation.
The DHCR7 specific inhibitor BM15766 inhibits chlamydialgrowth only in the absence of extracellular cholesterol
4-PI induced growth inhibition can be restored afterremoving the inhibitor or adding exogenous cholesterol
Figure 3 - C. trachomatis infected cells with or without 1mM 4-PI were incubated for 24hrs and the medium exchangedwith inhibitor, with inhibitor and 25ug/ml cholesterol, or medium without inhibitor and further incubated for additional24hrs. A) Cells were fixed at 24h or 48h p.i. Representative images (200x magnification fluorescence) are shown.B) Cells were harvested 24h or 48h p.i. and HeLa cells fresh infected with dilutions to titrate the C. trachomatis IFU.All titrations were done in triplicate. Error bars represent standard deviation.
Figure 2 - The figure shows the enzyme targets (name in red) of 4-PI and BM15766 (left side) and their catalyzedreaction (right side). The inhibitor 4-PI targets Lanosterol-14� demethylase, which catalyzes the demethylation of24-dihydrolanosterol to 4,4-dimethylcholesta-8(9),14-dien-3beta-ol (target methyl group in red). The 7-dehydrocholesterol reductase specific inhibitor BM15766 blocks the reduction of one double bond in dehydrocholesterolto cholesterol (target double bond in red), the very last step in the biosynthesis pathway.
The IDO-inhibitor 4-phenyl imidazole inhibitsC. trachomatis growth independent of IFN-�.
Figure 1 - Untreated (upper panel) and 50ng/ml IFN-� treated (lower panel) cells in the absence or presence of the1uM L-1-MT, 100uM and 1mM 4-PI were infected after 24hrs with an MOI of 1. After 24h p.i. cells were fixed inmethanol and stained with an FITC-conjugated antibody to C. trachomatis D than counterstained with Evans blue.Representative images (200x magnification fluorescence) are shown.
AcknowledgementThis work was supported by the National Institutes of Health gra nt AI 19782.
For further information: Contact Dr. Gerald Byrne: [email protected]
Simplified cholesterol biosynthesis pathwayin eukaryotic cells
ConclusionThese results suggest that CT149 has carboxylic esterase activity and contributes to thehydrolysis of eukaryotic cholesteryl esters during intracellular growth.
Our data suggest that CT149 plays a role in cholesterol metabolism by Chlamydia and maybe a target for chemotherapeutic development.
Future directionSubstrate speci�city of CT149 will be tested by using di�erent cholesteryl esters as well asother lipids including tri-, di, and monoglycerides
We will investigate if the inhibition of chlamydial growth is a result of the interaction of 4-Phenyl imidazole with CT149 or if other enzymes are involved.
Monoclonal antibodies against CT149 will be produced and used to observe the location andexpression within the host cell during the chlamydial infection to di�erent time points in thedevelopmental cycle.
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