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, LAbrami, 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: gbyrne@uthsc.edu
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.
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