Functional importance for developmental regulation of sterol biosynthesis in Acanthamoeba castellanii

Article abstract of DOI:10.1016/j.bbalip.2018.07.004

The sterol metabolome of Acanthamoeba castellanii (Ac) yielded 25 sterols. Substrate screening of cloned AcCYP51 revealed obtusifoliol as the natural substrate which converts to ?^8,14-sterol (<95%). The combination of [²H₃-methyl]methionine incubation to intact cultures showing C₂₈-ergosterol incorporates 2-²H atoms and C₂₉-7-dehydroporiferasterol incorporates 5 ²H-atoms, the natural distribution of sterols, CYP51 and previously published sterol methyltransferase (SMT) data indicate separate ?²⁴⁽²⁸⁾- and ?²⁵⁽²⁷⁾-olefin pathways to C₂₈- and C₂₉-sterol products from the protosterol cycloartenol. In cell-based culture, we observed a marked change in sterol compositions during the growth and encystment phases monitored microscopically and by trypan blue staining; trophozoites possess C₂₈/C₂₉-?^5,7-sterols, viable encysted cells (mature cyst) possess mostly C₂₉-?⁵-sterol and non-viable encysted cells possess C₂₈/C₂₉-?^5,7-sterols that turnover variably from stress to 6-methyl aromatic sterols associated with changed membrane fluidity affording lysis. An incompatible fit of steroidal aromatics in membranes was confirmed using the yeast sterol auxotroph GL7. Only viable cysts, including those treated with inhibitor, can excyst into trophozoites. 25-Azacycloartanol or voriconazole that target SMT and CYP51, respectively, are potent enzyme inhibitors in the nanomolar range against the cloned enzymes and amoeba cells. At minimum amoebicidal concentration of inhibitor amoeboid cells rapidly convert to encysted cells unable to excyst. The correlation between stage-specific sterol compositions and the physiological effects of ergosterol biosynthesis inhibitors suggests that amoeba fitness is controlled mainly by developmentally-regulated changes in the phytosterol B-ring; paired interference in the ?^5,7-sterol biosynthesis (to ?^5,7) - metabolism (to ?⁵ or 6-methyl aromatic) congruence during cell proliferation and encystment could be a source of therapeutic intervention for Acanthamoeba infections.

Full text of DOI:10.1016/j.bbalip.2018.07.004

Accepted Manuscript
Functional importance for developmental regulation of sterol
biosynthesis in Acanthamoeba castellanii
Wenxu Zhou, Andrew G.S. Warrilow, Crista D. Thomas, Emilio
Ramos, Josie E. Parker, Claire L. Price, Boden H. Vanderloop,
Paxtyn M. Fisher, Michael D. Loftis, Diane E. Kelly, Steven L.
Kelly, W. David Nes
PII:
S1388-1981(18)30169-0
doi:10.1016/j.bbalip.2018.07.004
BBAMCB 58329
DOI:
Reference:
To appear in:
BBA - Molecular and Cell Biology of Lipids
Received date:
Revised date:
Accepted date:
23 March 2018
26 June 2018
20 July 2018
Please cite this article as: Wenxu Zhou, Andrew G.S. Warrilow, Crista D. Thomas, Emilio
Ramos, Josie E. Parker, Claire L. Price, Boden H. Vanderloop, Paxtyn M. Fisher, Michael
D. Loftis, Diane E. Kelly, Steven L. Kelly, W. David Nes , Functional importance for
developmental regulation of sterol biosynthesis in Acanthamoeba castellanii. Bbamcb
(2018), doi:10.1016/j.bbalip.2018.07.004
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ACCEPTED MANUSCRIPT
Classification: Biological Sciences, Biochemistry
Functional Importance for Developmental Regulation of Sterol Biosynthesis in
Acanthamoeba castellanii
1
±
2±
1±
1
2.
Wenxu Zhou , Andrew G.S. Warrilow, Crista D. Thomas , Emilio Ramos, Josie E. Parker,
2
1
1
1
2
Claire L. Price, Boden H. Vanderloop, Paxtyn M. Fisher, Michael D. Loftis, Diane E. Kelly,
2
1*
Steven L. Kelly , and W. David Nes
1
Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas, United
States of America
2
Center for Cytochrome P450 Biodiversity, Institute of Life Science, College of Medicine,
Swansea University, Swansea, Wales, United Kingdom
*Corresponding author: W. David Nes, Department of Chemistry & Biochemistry, Texas Tech
University, Lubbock, Texas, USA 79424, E-mail: wdavid.nes@ttu.edu (WDN)
±
These authors contributed equally to this work.
1

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Abstract
The sterol metabolome of Acanthamoeba castellanii (Ac) yielded 25 sterols. Substrate screening
8
,14
of cloned AcCYP51 revealed obtusifoliol as the natural substrate which converts to ∆ -sterol
2
(
<95%). The combination of [ H -methyl]methionine incubation to intact cultures showing C -
3
2
8
2
2
ergosterol incorporates 2- H atoms and C -7-dehydroporiferasterol incorporates 5 H-atoms, the
29
natural distribution of sterols, CYP51 and previously published sterol methyltransferase (SMT)
2
4(28)
25(27)
data indicate separate ∆
- and ∆
-olefin pathways to C - and C - sterol products from
28 29
the protosterol cycloartenol. In cell-based culture, we observed a marked change in sterol
compositions during the growth and encystment phases monitored microscopically and by trypan
5
,7
blue staining; trophozoites possess C /C -∆ -sterols, viable encysted cells (mature cyst)
2
8
29
5
5,7
possess mostly C -∆ -sterol and non-viable encysted cells possess C /C -∆ -sterols that
2
9
28 29
turnover variably from stress to 6-methyl aromatic sterols associated with changed membrane
fluidity affording lysis. An incompatible fit of steroidal aromatics in membranes was confirmed
using the yeast sterol auxotroph GL7. Only viable cysts, including those treated with inhibitor,
can excyst into trophozoites. 25-Azacycloartanol or voriconazole that target SMT and CYP51,
respectively, are potent enzyme inhibitors in the nanomolar range against the cloned enzymes
and amoeba cells. At minimum amoebicidal concentration of inhibitor amoeboid cells rapidly
convert to encysted cells unable to excyst. The correlation between stage-specific sterol
compositions and the physiological effects of ergosterol biosynthesis inhibitors suggests that
amoeba fitness is controlled mainly by developmentally-regulated changes in the phytosterol B-
5
,7
5,7
5
ring; paired interference in the ∆ -sterol biosynthesis (to ∆ ) - metabolism (to ∆ or 6-methyl
aromatic) congruence during cell proliferation and encystment could be a source of therapeutic
intervention for Acanthamoeba infections
Key words: Acanthamoeba castellanii, sterol methyltransferase, sterol evolution, aromatic
sterols, trophozoite, encystment, ergosterol biosynthesis inhibitor
Abbreviations: Ac, Acanthamoeba castellanii, SMT, sterol methyl transferase, GC-MS, gas-
chromatography-mass spectroscopy, VOR, voriconazole, AZC, 25-azacycloartanol, RRTc,
retention time of unknown to retention time of cholesterol in GC. Mya, million years ago.
Highlights:
1
. Acanthamoeba castellanii cells generate 25 sterols, a mixture of 4-methyl intermediates
5
and ∆ -final products, affording a biosynthetic pathway recurrent from an ancient algal
2
4(28)
25(27)
predecessor; separate intermediates via the ∆
-methyl olefin route and ∆
- ethyl
olefin route determine the C - and C -sterol balance in the amoeba.
2
8
29
2
3
. Cloned AcCYP51 favors obtusifoliol as substrate, but can convert cycloeucalenol weakly
1
4
to ∆ -product.
5
,7
5
. ∆ - and ∆ -sterols are differentially correlated to trophozoite growth and mature cyst
viability and excystment, respectively, while 6-methyl aromatic sterols are a chemical
signature indicative of encysted cells on the path to death.
4
. Voriconazole inhibits AcCYP51 as other azoles and 25-azacycloartanol inhibits AcSMT
in the low nanmolar range; these drugs are potent inhibitors of trophozoite growth and
impair encystment toward viable cyst production in the range of 500 nM to 3 µM.
2

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1
. Introduction
Sterol synthesis is a basic metabolic pathway of eukaryotes giving rise to essential membrane
components. The biosynthesis pathway is typically divided into three routes, two of which
characterize the phytosterol biosynthesis pathways in land plants and fungi yielding C -
2
9
sitosterol (24α-ethyl group) and C -ergosterol (24β-methyl group), respectively, and in animals
2
8
yielding C -cholesterol (Fig. 1) [1-4]. These contemporary biosynthetic pathways retain
2
7
ancestral features along with novelties specific to their particular lineages. Notably, the
protosterol product of the 2,3-oxidosqualene synthase yielding cycloartenol or lanosterol
associates phylogenetically to plant-animal divisions while the substrate acceptability and
5
mechanisms of individual enzymes catalyzing completion of the pathway to ∆ -sterols reveal
relatedness or divergence in catalytic competence across kingdoms [5, 6]. For phytosterols
generating diversity, the size, location and direction (α- or β-orientation) of the side chain C24-
alkyl group correlate to plant-fungal divisions or to more- or less-advanced in descent. Structural
features common to sterols are a cyclopentanoperhydrophenanthrene ring system, polar C3-OH
group and intact side chain of 8 to 10-carbon atoms. These characteristics contribute positively to
the molecule’s overall flat shape, length and amphipathic character relevant to the architectural
suitability of sterols in membranes [7]. Alternatively, there can be harmful features in
intermediates that are removed during the normal course of metabolism. For example, the C4-
5
geminal methyl groups and C14-methyl group on the protosterol, eliminated to yield ∆ -sterols,
are known to affect the hydrogen bonding strength of the C3-OH group and planarity of the α-
(back) face of the sterol nucleus, respectively; when intermediates that possess these features
accumulate in the cell the result can be detrimental to sterol homeostasis that thereby, interferes
with growth and maturation [8-10].
Despite the prevailing hypothesis suggesting progressive structural modifications in the
protosterol formed by prokaryotes parallels a gradual cholesterol evolution in response to
increases in the atmospheric concentration of oxygen [11-13] recent chemical analysis on the
5
origin of these compounds indicate a complete sterol biosynthesis to ∆ -products may have
existed in the most primitive eukaryotes, evidenced in the presence of cholestane and its C28- to
C30-carbon homologs in molecular fossils found in rocks and formations between 750 Mya to
1
,000 Mya [14]. Additionally, phylogenomic analysis shows the genes of 13 sterolic enzymes
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yielding these steranes could be codified in the genomic organization of the last eukaryotic
common ancestor (LECA) approximately 2.45-2.32 Mya [15, 16] with orthologous prokaryotic
enzymes likely formed during the Great Oxidation Event by horizontal gene transfers [17, 18].
Based on this new information, we surmise competing phylogenies constructed from these
characters could reasonably describe the punctuated and recurrent evolution of sterol
biosynthesis to amoeba ergosterol deeply rooted in primary metabolism of the LECA [19, 20].
However, relevant to the understanding of sterol biosynthesis in Acanthamoeba for the purpose
of inhibition, the phyla-specific canonical routes to alternate sterol products must also be
considered, because different steps in sterol metabolism – sterol side chain or nucleus revisions-
could interface uniquely through stage-specific structural adjustment during the amoeba life
history, as reported can occur in land plants [21, 22] to yield ergosterol convergence (Fig. 1).
Acanthamoeba spp. has a number of particularly advantageous characteristics for research on
sterol function and evolution. In the first place, the differentiation process originating in the
trophozoite and passing through a pseudocyst, i.e., an intermediate structure composed of a wall
that may not protect against adverse conditions [23, 24], can yield multiple cyst types, including
1
) viable cysts with a resistant double-layered outer wrinkled ectocyst and inner endocyst and 2)
a mixture of non-viable cysts of apoptotic cells that possess a fragile cell wall and dead cells
generated from autophagocytosis [25]. These studies further show azolic inhibitors-voriconazole
can prevent trophozoite growth as well as activate an encystment pathway with a final trajectory
ending in programed cell death [26]. Ergosterol biosynthesis inhibitors with various structures
can therefore be administered and the effects on growth and differentiation measured in
correlation to changes in sterol biosynthesis. Intriguingly, the unikont Acanthamoeba is distinctly
different from fungi and bikont protozoa (kinetoplastids) in their sterol biosynthesis capabilities.
The variant pathways for C -ergosterol biosynthesis in parasitic protozoa also differ from the
2
8
C -cholesterol biosynthesis in the human host where sterol C24-methylation is lost [27].
27
5
The ability of Acathamoeba spp to synthesize a plant-based pathway of cycloartenol to ∆ - and
5
,7
∆
-24β-C - and C -alkyl sterols was discovered in the early 1980’s by Raedorstorff and
28 29
Rohmer [28, 29]. These researchers together with the Korn group demonstrated the amoeba
ergosterol and 7-dehydroporiferasterol can convert to uncommon C - and C -phenanthrene 6-
2
8
29
methyl aromatic sterols [30, 31]. The Acanthamoeba system has been exploited in the
4

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collaborative laboratories of Kelly and Nes [32, 33] and more recently by Roberts and coworkers
[
34]. However, unlike the Rohmer and Korn groups, the more recent authors [34] have
concluded that Acanthamoeba operates a fungal-based pathway of lanosterol to ergosterol and
the protozoan is unable to synthesize C -sterols. Our own preliminary work corroborated the
29
original findings, and the purpose of the present paper is to document this in detail and to expand
the study to encompass a more comprehensive evaluation of the biosynthetic enzymes and
steroidal nuggets expressed variably at stage-specific times in Acanthamoeba. The approach used
in our investigation has been chosen to validate the ergosterol biosynthesis pathway from
2
5(27)
cycloartenol and the existence of a new ∆
-C -sterol biosynthesis pathway in amoeba that
29
recapitulates the green algal sterol biosynthesis pathway to C -sterols.
2
9
Another reason for choosing Acanthamoeba was the critical factors in target deconvolution using
sterolomics, bioinformatics and genetic technologies that led to enzyme-based strategies for
manipulating the ergosterol biosynthesis pathway in this and related parasitic protozoa. These
various investigations provide new chemical starting points against our target enzymes- the
Acanthamoeba castellanii (Ac) sterol C14-demethylase (CYP51) and sterol C24/C28-
methyltransferases (SMT). Inhibition of either enzyme by a rationally designed tight binding
inhibitor rapidly yields trophozoite death [33]. The only real uncertainties left about the
importance of phytosterol biosynthesis/metabolism in Acanthamoeba are: (i) can the sterol
composition change during the amoeba life cycle, (ii) has phylogenetics influenced formation of
separate routes to C - and C -sterols, (iii) to what extent is ergosterol biosynthesis during the
2
8
29
Acanthamoeba encystment-excystment cycle vulnerable to inhibitors and (iv) what role is played
5
,7
5
in Acanthamoeba physiology by changing the sterol B-ring structure from ∆ - to ∆ - or 6-
methyl aromatic group?
In this study, the pattern of sterol biosynthesis and accumulation in Acanthamoeba against
ultrastructure and viability of the cells was determined at various stages of amoeba proliferation
and encystment. In addition, we examined the physiological role of the uncommon 6-methyl
aromatic sterol structures generated by the amobea through use of enzyme inhibitors and the
yeast sterol auxotroph GL7. The results define the biosynthetic factors that control the changes in
sterol compositions associated with amoeba differentiation, and provide the necessary foundation
for further regulatory studies at the biochemical and molecular levels. Given the therapeutic
5

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importance for CYP51 and SMT as chokepoint enzymes in Acanthamoeba and other parasitic
protozoa, knowledge of the diversity and function of terminal C - and C -sterols in the
2
8
29
trophozoite and encysted cell can be of value in developing new inhibitor chemotypes to treat
amoeba diseases.
6

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2
. Materials and methods
2
.1. Strain and culture conditions. Acanthamoeba castellanii strain ATCC 30010 was inoculated
into tissue culture 25 ml T-flasks prepared with ATTC media 712. Cells were cultured axenically
0
in 5 ml static culture at 25 C with occasional hand shaking to keep cells in suspension.
Continuous cultures were maintained by subculture of 4-5 day growth arrested cells into fresh
medium. Growth experiments to establish the growth curve and for sterol analyses were typically
4
carried out using 1 x 10 trophozoites (< 90%) or cysts derived by the methods below. The
number of individual amoeba distinct from encysted cells that possess a double wall per ml was
determined microscopically. For cell aggregates a best effort was made to count the number of
individual encysted cells in the clump. Morphological changes and encystation ratios were
calculated by counting trophozoite to cysts using a Neubauer hemocytometer under an Olympus
CH-2 compound light microscope and in separate observations cell differentiation was detected
with an inverted microscope monitored daily for 2 weeks. Cells were pelleted by centrifugation
for 5 min at 5,000 g. Cell viability was checked by trypan blue exclusion method.
2
.2. Induction of encystment. To generate viable cysts capable of excystment, equivalent to
resting or dormant cysts, three different approaches were employed. Method-1, using wild-type
trophozoites, encystment was induced naturally by allowing the early growth arrested cultures
(ca. 4-5 day synchronous cultures) to deplete essential growth nutrients required in metabolite
biosynthesis. In a second approach encystment was forced by two replacement methods.
Method-2 is salt- induced encystment and was used by Korn et al. and Mehdi and Garg [24, 30,
3
5]. Briefly, trophozoites were resuspended in medium containing 1 mM MgCl to a cell
2
6
0
concentration of approximately 6.5 x 10 cells/ml. The culture was incubated in a 30 C shaker at
50 rpm for 2 days. The cells were collected and treated with 1% sodium dodecyl sulfate (SDS)
1
solution for 30 min at room temperature to remove trophozoites, fragile encysted cells and cell
debris. The resulting SDS-resistant cysts were washed with PAGE’s saline solution 5 times and
analyzed for sterols. Method 3 is starvation-induced encystment where the amoebae are
maintained on an agar-embedment system for 2-4 weeks [23]. Encystment synchrony was
evaluated morphologically using a combination of inverted microscopy, light microscopy and
electron microscopy. More than 90-95% of the cells formed from these conditions yielded
encysted cells with features representative of resting or dormant cysts. Resting cysts generated
7

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5
by the natural and replacement methods were determined viable by their ∆ -sterol profile, and in
each case yielded similar excystment activities of about 100%. Typically, the viable cysts at the
end of these experiments appeared as single cells in light microscopy either on the solid or in
liquid medium. A third approach was used to promote encystment by administering drugs to the
medium.
2
.3. Trophozoite toxicity assay. To determine the effect of the test compounds on the growth of
5
Acanthamoeba, trophozoites, approximately 1 x 10 cells, were inoculated into 24 well (4 x 6)
microtiter plates of 3 ml final volume taking the inoculum from cells grown in the T-flask
continuous culture system. Stock inhibitor concentrations were prepared in dimethylsulfoxide
(not to exceed 1% solvent) and as relevant serially diluted to final concentrations in wells that
ranged from 16 nM to 30 µM (approximate to the end point for inhibitor studies administered to
human epithelial kidney cells [32, 33]. Control wells containing medium and 1% DMSO solvent
0
had no effect on the cell growth. The microliter plates were incubated at 25- C for 48 hr and the
resulting number of trophozoites to cysts counted at the end of the incubation using the
hemocytometer. The percentage of viable tophozoites following to different concentrations of
inhibitors was determined by the trypan blue exclusion method. The 50% effective does (IC )
5
0
value of inhibitors against tophozoite growth under the stated assay conditions was determined
by linear extrapolation using GraphPad Prism (GraphPad Software Inc., CA). Cells stained blue
were considered non-viable while live cells were unstained. These experiments were carried out
in triplicate and at different time points. The drug concentration responsible for the minimum
ameobacidal activity (MAC) was defined as the lowest concentration of the inhibitor with no
visible live trophozoites, as determined by visual inspection of the treated cultures using the light
microscope following trypan blue staining and inverted microscope which confirmed cell death
by the absence of trophozoite cells and notable accumulation of cell debris. Observations were
performed in triplicate x 2 using light microscope for counting cells. There was excellent
agreement (≤10% variation for any data point) in the growth response to inhibitors in the two
independent trials.
2
.4. Time-dependent killing. A log-phase culture of Trophozoites was seeded into 24-well plates
as described above. Cells were then challenged with inhibitors at previously determined MAC
concentrations. At 12 hr intervals, aliquots were removed and counted and cell viability
8

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determined; at 48 hr remaining cells were pelleted and resuspended into fresh medium and
allowed to incubate for another 5 days and the cells counted to determine trophozoite
proliferation and analyzed for sterol content. Experiments were performed with biological
replicates. A metabolic marker was used to assess viable cells by means of GC-MS analysis.
Viable trophozioites were associated with the production of ergosterol/7-dehydroporiferaterol
and viable cysts were associated with poriferasterol production and cells in the death mode were
associated with steroidal aromatics as discussed in the text.
2
.5. Cysticidal assay. Cysts, generated by the methods above, or derived from inhibitor
treatment, were incubated with the anti-amoeba drug under the same assay conditions described
for ameobacidal assay. Growth was defined as the presence of excysted trophozoites found
growing in 24 well plates or 5 ml T-flasks after 5 days of growth.
2
.6. Chemical and instrumental analysis. The source of sterol substrates and steroidal inhibitors
evaluated in this study is described in earlier papers [8, 20, 36]. Voriconazole was purchased
from Sigma. All sterols were purified by HPLC to < 95% by GC analysis. S-adenosyl-L-
3
methionine (SAM) chloride salt was purchased from Sigma, [methyl- H ]SAM (specific activity
3
2
1
0-15 Ci/mMol, and diluted to 10 µCi/µMol, tetraosylate (methyl- H ]SAM (99% atom
3
2
enrichment) purchased from S/D/N isotopes (Pointe-Claire, QC), L-[methyl- H ]methionine
3
(Sigma). The Bradford protein assay kit was purchased from Bio-Rad and isopropyl-1-thio-β-D-
galactoside (IPTG) purchased from Research Products International Corp. DNA synthesis
inhibitor 5-fluor-2-deoxyuridine (FUdR) was purchased from MP Biomedicals LLC. All other
reagents and chemical were from Sigma or Fisher unless otherwise noted.
1
Instrumental methods have been reported previously [19, 20]. Briefly, H-NMR spectra were
recorded in CDL at ambient temperature using a Varian Unity Inova 500 MHz spectrometer;
3
chemical shifts (δ, ppm) are referenced to chloroform, (δ, 7.26 ppm). Mass spectra were obtained
on a Hewlett-Packard 6890 GC-HP 5973 MSD instrument (electron impact, 70eV, scan range
5
0-550 amu). HPLC was carried out at room temperature using a Phenomenex Luna C18-column
(250 mm x 4.6. mm x 5 µM) connected to a diode array multiple wavelength diode array detector
with methanol as eluent. Capillary GC (0.25 mm i.d, by 30 m fused silica column coated with
Zebron ZB-5 from Phenomenex) was operated at a flow rate of He set at 1.2 ml/min, injector
0
port at 250 0C, and temperature program of initial 170 C, held for 1 min, and increased at 20
9

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0
0
C/min to 280 C. GC analysis of sterols is reported as RRTc values referring to the retention
time of sample GC peak to retention time of cholesterol peak which moved typically in the
chromatogram at 13.8 min or slightly different to 14.5 min depending on whether the column tip
was clipped due to age issues. In HPLC samples were separated on a Phenomenex Luna C -
1
8
column (250 mm x 4.6 mm x 5 cm) connected to a diode array multiple detector with samples
eluted with methanol; the αc of the sterol is the elution time of compound relative to the elution
time of cholesterol, which is 20.6 min. In sterol analysis, product distributions were determined
by approximate integration of chromatographic peaks, experiments were performed in biological
replicates with excellent agreement between trials (>95%). Thin layer chromatography was
performed on 10 x 20 cm, 250 µM silica plates (Baker) developed twice in benzene-ether (85/15,
v/v).
2
.7. Cell metabolite identification. Typically, small-scale amoeba cultures were harvested
different points along the growth curve in the presence and absence of steroidal inhibitor and
azole at MAC concentrations of the compound. Control or treated cultures in 5 ml or 10 ml
medium dispersed in 25 ml T-flasks were inoculated with trophozoites (90%) or resting cysts
(100%) and cultured for the desired time without shaking and sterol analyzed at the end of the
incubation period. Independently, trophozoites were incubated in the 24-well plate system
containing 1 ml total medium and after 48 h treatment the sterol composition determined by GC-
MS analysis. Cell pellets were split with an internal standard of 5α-cholestane added to one of
the cell pellets for determination of sterol amounts in cells. Cells were saponified with 10%
methanolic KOH extracted with hexanes and the neutral lipids routinely analyzed by GC-MS for
structure determination and quantification. Components were identified by comparison of
retention times in GC and HPLC and mass spectra with authentic standards in our own collection
[
19, 20, 22, 37] and were quantified by comparison of detector response with that of the internal
standard. As relevant preparative scale incubation were carried to obtain a subset of amoeba
1
sterols for HNMR analysis.
2
.8. Scanning (SEM) and transmission (TEM) electron microscopy. SEM: Prior to fixation,
control and treated cell pellets were rinsed in 10% phosphate buffer solution, and centrifuged
again to remove any salts that would give false images of crystalline-like structures in the
background. After resuspension in buffer, cells were adhered onto poly-L-lysine-coated glass
1
0

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coverslips and subsequently fixed with a solution of 2.5% glutaraldehyde in 0.05M cacodylate
buffer. Samples were postfixed for 10 min in 1.5% OsO4, dehydrated in ethanol, and critical
point dried with liquid CO . The cells were then coated with technics hummer V coater and
2
observed under a Zeiss 540 scanning electron microscope.
TEM: Similar to SEM, prior to fixation, control and treated cell pellets, that included primarily
trophozoites or cysts depending on the treatment, were rinsed in 10% phosphate buffer solution,
and centrifuged again. After resuspension in buffer, cells were adhered onto poly-L-lysine-
coated glass coverslips and subsequently fixed in a solution of 2.5% glutaraldehyde in 0.05M
cacodylate buffer. After fixation, the cells were post-fixed with 1% (wt/vol) OsO4, dehydrated
with ethanol and acetone and embedded in epoxy resin. Ultrathin sections (1 micron) were
contrast stained with uranyl acetate and lead acetate and observed under a Hitahci H-8100
Transmission Electron Microscope fitted with AMT side mount digital camera.
2
.9. Induction of aromatic sterol synthesis. Following the protocol of Korn [30] enabling the
rapid induction of aromatase activity through cell disruption coupled to osmotic stress, control
7
amoeba cell pellets harboring approximate 1 x 10 cells/ml composed mostly of trophozoites
(
90%) were equilibrated for 20 min at zero degrees in 0.002 M CaCl -0.001 M Tris buffer (pH
2
7
.0) and homogenized with three gentle strokes of a Douce homogenizer. Samples of 20 ml were
incubated at 18 degrees for 6 h. At the end of the incubations, the lipids extracted with
chloroform-methanol (2:1. v/v). The solvent was evaporated and the resulting organic layer
analyzed by GC-MS/HPLC-UV or in separate study, preparative scale studies were carried out to
1
isolate the major sterol products for HNMR.
2
.10. Determination of AcCYP51 substrate affinity and product outcome
+
The expression of pCWori :AcCYP51 construct in E. coli has been described previously [32].
Cytochrome P450 concentration, determined by reduced carbon monoxide difference spectra,
and ligand binding studies [6, 38]. The dissociation constant of the enzyme-ligand complex (Kd)
for each sterol or azole was determined by non-linear regression (Levenberg-Marquardt
algorithm) using a rearrangement of the Morrison equation for tight ligand binding and the
Michaelis-Menten equation when ligand binding was no longer tight [38]. Stock 2.5 mM sterol
solutions were prepared in 40% (w/v) of 2-hydroxypropyl-b-cyclodextrin (HPCD). All spectral
1
1

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determinations were determined in triplicate. Curve-fitting of ligand binding data were
performed using the computer program ProFit 6.1.12 (QuantumSoft, Zurich, Switzerland).
Spectral determinations were made using quartz semi-micro cuvettes with a Hitachi-U 3310
UV/Vis spectrometer (San Jose, CA). CYP51 enzyme activity from 10 min incubation was
determined in triplicate using a CYP51 reconstitution assay system (0.5 ml final volume)
composed of 1.14 µM AcCYP51, 2.81 µM Aspergillus fumigatus (AfCPR1-Q4WM67)
cytochrome P450 reductase, 50 mM sterol substrate, 50 mM dilauryl phosphatidylcholine, 4%
-1
HPCD, 0.4 mg ml isocitrate dehydrogenase, 25 mM trisodium isocitrate, 50 mM NaCl, 5 mM
MgCl and 40 mM MOPS (pH, 7.2). In a preliminary study to establish approximate K and K
cat
2
m
kinetic constants for obtusifoliol against pure Ac C14-demethylase, reactions were carried out at
0
3
7 C with 1.14 µM recombinant CYP51 and 2.74 µM Homo sapiens CPR (as redox partner) at
sterol concentrations of 6.25, 12.5 25, 50, 75 and 100 µM for 20 min. In a separate set of
experiments, the kcat for AcCYP51 against AfCPR1 was determined under assay conditions
performed with the HsCPR redox partner. Because the turnover number for obtusifoliol was
distinctly low against HsCPR redox partner we chose to evaluate sterol structure-activity against
the redox partner of AfCPR. Enzyme-generated products were recovered by methanolic KOH
saponification followed by extraction with 2 x 3 ml n-hexane, and then evaporation to dryness
using a vacuum centrifuge. Product outcome was determined by GC-MS analysis of the free
sterol. Competition experiments to establish IC of azole against obtusifoliol were carried with
5
0
the AcCYP51 and redox partner HsCPR.
2
.11. AcSMT Inhibition studies
Enzymatic assay conditions with soluble (cloned) Ac24-SMT or Ac28-SMT enzyme preparation
and inhibition constant (IC ) determination against natural substrate and the 25-azacycloartanol
5
0
inhibitor were essentially as described in the previous report [33]. Briefly, IC50 values were
determined from a dose-response curve against variable inhibitor concentrations in the range 0
nM, 3.9 nM to 1 µM in the presence of fixed-saturation- concentration of sterol acceptor (100
µM) and coenzyme (150 µM). Assays were performed in triplicate with less than 10% deviation.
IC50 values were determined by standard graphical procedures for which computer-assisted
linear regression analysis afforded correlation coefficients greater than 0.95 in a cases.
Conversion of IC value to K as dissociation constant, based on the experimentally deduced
5
0
i
1
2

ACCEPTED MANUSCRIPT
kinetic properties of cycloartenol (24-SMT) or 24(28)-methylene lophenol (28-SMT) and 25-
azacyclartanol was accomplished using the Cheng-Prussoff equation [38].
2
.12. GL7 growth
Sterol structure-membrane response effects were carried out using the Saccharomyces
cerevisiae strain GL7 with a defective 2,3-oxidosqualene to lanosterol synthase (LAS)
auxotrophic for ergosterol, its natural membrane insert [8, 39]. GL7 was cultured on 5 mg/l and
Tween 80 (15 ml/l) in yeast-peptone-dextrose medium for 72 h affording growth arrest typical of
8
wild-type yeast of 1 x 10 cells/ml [8].
1
3

ACCEPTED MANUSCRIPT
RESULTS
.1 Development of novel steroidogenesis patterns with growth and differentiation.
3
A single cohort of viable cysts cultured on optimal growth conditions linked to extrinsic cues of
salt stress or a decrease in carbon sources [23, 24] was used to observe excystment to replicative
senescence-death and any associated sterol patterning. The initial experiments to observe
5
differentiation included inoculum obtained from cysts determined to possess exlcusively∆ -
4
sterols - brassicasterol and poriferasterol. Trophozoite proliferation, from 10 resting cysts/ml,
followed a logarithmic course with a doubling time of 2.8 h. There seemed to be a slight
induction period related to excystment phase of approximate 8-12 h yielding trophozoites.
Amoeba cell proliferation reached growth arrest at approximately 3 days affording a cell density
1
40 thousand cells/ml (Fig. 2, Panel A). As the amoeba continued in stationary phase, they began
to progressively convert to encysted cells of mixed population of live and dead. The total cell
number decreased with culture age as did the fresh weight of cultures by roughly a factor 10 at
day 7 (Fig. 2, Panel B-inset). Continued incubation of the encysted cells for a month led to dead
cells by trypan blue staining and to further decrease in the number of encysted cells by another
4
factor of ten to about 10 /ml cysts remaining. Because some media preparations for amoeba
growth include fetal bovine serum (FBS), which we determined contains 10µM cholesterol, we
added FBS as described in the American Type Culture Collection growth media or added 10µM
cholesterol in DMSO to our growth media. In either case, the cholesterol supplement had only a
minor stimulation effect on trophozoite growth.
To assess the conservation or change in sterol composition associated with differentiation, cells
were harvested at 6 different stages in the excystment-trophozoite-encystment cycle at days 1, 2,
3
, 4, 7 and 1 month post-inoculation. The analysis demonstrated developmental changes in cell
morphology and viability were accompanied by remarkable changes in steroidogenesis and
metabolism. First, in what may be considered the amoeba growth phase, in the early logarithmic
phase of growth in days 1 to 1.5 the C and C -cyclopropyl sterols -cycloartenol and 24(28)-
3
0
31
methylene cycloartanol accumulate (Fig 2 and Suppl. Fig. 1), followed in logarithmic growth in
5
,7
days 2 to 3/3.5 by a shift to ∆ -C - and C -sterol end product formation in approximate 5 to 7
2
8
29
ratio (Fig. 2, Panel B and Suppl. Fig. 2).
1
4

ACCEPTED MANUSCRIPT
At day 4 where the total cell number was similar to that at day 3, there was a mixed population
of trophozpoite and cysts and the sterol composition of the pelleted cells was a mixture of
ergosterol, 7-dehydroporiferasterol, brassicasterol and poriferasterol roughly in a 50:50 ratio of
5
,7
5
∆
to ∆ -sterols. At day 7, most trophozoites had encysted (90% of total cells). The fresh weight
of the cell pellet had decreased by a factor of 3 and the sterol composition of the encysted cells
5
had reverted back to that of inoculum of ∆ - C -sterol (90% of total sterol). (Figure 2, Panel B
2
9
inset). At one-month incubation, after the remaining encysted cells had opportunity to excyst and
proceed to trophozoite and then encyst again in cyclic fashion using up the carbon sources, most
encysted cells were dead and contained predominantly C - and C - 6-methyl aromatic sterols,
2
8
29
which we refer to as amebasterol-1 and amebasterol-2, respectively. These sterols were
previously observed by Korn and Rohmer in Acanthamoeba as minor compounds (trace to 5%
total sterol) [28-30, 40] from growth arrested cultures. We confirmed the structure of the major
Ac sterols, as well as that of brassicasterol which is a newly characterized Ac sterol, through an
examination of their chromatographic and spectral properties reported in Figure 3 (Suppl. Table
1
1
for HNMR analysis).
With this knowledge, the morphology and ultrastructure characteristics of trophozoites and
encysted cells obtained from control (and subsequently treated cells) were determined. In light
microscopy, inoculum cells generated by Method 2 against SDS (Materials and Methods) were
found to possess the double-wall feature, and in transmission electron microscopy the
encystment was evidenced in ultrastructure showing a nucleolus with a dense droplet in the cyst
center, dense cytoplasm packed with lipid droplets, spherical mitochondria (M) and
autolysosomes (A) that appear as large basophilic granules. Our observations, in agreement with
Bowers and Korn [24], showed the mean diameter of rounded amoeboid cells were
approximately 26.5 ± 0.17 µ whereas the mean cytoplasmic diameter of cysts was 16.2 ± 0.13 µ.
The decrease in surface area during encystment of a cell of median diameter was more than 65%
while the decrease in median cell volume was approximately 80%, suggesting significant loss of
cell membrane from amoeba to true cyst. The sterol content in trophozoite and true cyst was 12
pg/cell and 4 pg/cell, respectively. Thus, there is both a distinct loss in total sterol and a marked
5
,7
metabolism of preformed ∆ -sterol as trophozoite converts into the true cyst form. These sterol
modifications correlate to the different membrane requirements of non-encysted and encysted
cells capable of excystment (Figure 2). Once true cysts differentiate into trophozoites, the
1
5

ACCEPTED MANUSCRIPT
resulting amoeba, observed to synthesize ergosterol and 7-dehydroporiferaterol, were examined
by light and electron microscopy. These non-encysted amoebae were shown to contain typical
cytoplasmic organelles, such as a nucleus with a prominent nucleolus, an abundant rough
endoplasmic reticulum, mitochondria, vacuoles and lipid droplets and showed surface
protrusions -acanthapodia, durable double wall of smooth ectocyst and a round endocyst (Fig. 2,
Panel C).
3
.2. Medium-replacement induced-encystment can lead to distinct cell types of characteristic
sterol compositions.
5
To determine whether the unusual ∆ -sterol and 6-methyl aromatic sterol profiles observed in the
stationary phase growth were associated with distinct cyst populations, we initiated encystment
by Method 2. The salt stressed trophozoite cultures spontaneously encysted yielding some cell
debris. GC-MS analysis of the pellet extract revealed 21 sterols (Suppl. Fig. 2, Panel E and Table
1
) which is the most complex sterol composition reported for not only the amoeba but for
protozoa generally. Many of these sterols represent co-metabolites and by-products. Of interest
5
,7
was the experimentally induced encystment of amoeba yielded a sterol mixture of ∆ -sterols,
5
∆
-sterols and 6-methyl aromatic sterols. Notably, no significant trpohozoites were detected
microscopically, yet there is a significant amount of ergosterol and 7-dehydroporiferasterol in the
5
,7
sterol mixture suggesting that a subset of encysted cells could possess ∆ -sterols. As the
encysted cells were processed further through SDS-treatment, the cells that remain contain
predominantly brassicasterol and poriferasterol by GC-MS analysis (Supple Fig. 2, Panel F);
these cells are viable and can excyst completely to trophozoites in enriched medium in 12 h. We
surmise the salt step can create mixed populations of encysted cells; one population is composed
5
,7
of fragile double walls and membranes composed of ∆ -sterols or aromatic sterols and a second
5
population is composed of resistant double-wall structures and membranes composed of ∆ -
sterols.
A crucial observation made by the Korn group to prepare an abundance of 6-methyl aromatic
sterols of the phenanthrene skeleton was to generate a cell-free system of Ac in high salt solution
yielding a microsomal (membranes) subcellular fraction. Under these conditions, the ergosterol
and 7-dehydroporiferasterol rapidly convert in stoichiometric fashion to amebasterol-1 and
amebasterol-2; the aromatic compounds remain membrane-bound [30]. The relevance of the
1
6

ACCEPTED MANUSCRIPT
Korn study is to show both that the phenanthrene steroids can be easily obtained in amounts
necessary for structure determination and that these aromatic compounds can associate with the
endoplasmic-plasma-membrane fraction of microsomes in intact cells. Notably, this approach
used by Rohmer on the related Acanthamoeba species-polyphaga yielded sufficient compound to
1
3
1
be characterized by C/ H-NMR to prove the structure and stereochemistry of the metabolites as
new class of steroids [31]. We repeated these studies and observed Ac homogenates generate 6-
5
,7
methyl aromatic sterols from ∆ -sterols in amounts reported by Korn over the 6 h incubation
period. The 6-methyl aromatic sterols can remain stable for at least 2 months (Suppl. Figure 2,
Panels G and H).
3
.3. Catalytic competence of AcCYP51relative to Ac24-SMT and Ac28-SMT.
+
The functional expression in E. coli of pC Wori :AcCYP51 construct and wild-type cDNA of 24-
SMT and 28-SMT have been described previously [32, 33]. To elucidate the diversity of
substrate preference that could influence the order of intermediates in C - and C - sterol
28
29
synthesis, eleven 14-methyl sterols were evaluated for substrate recognition by AcCYP51 (Table
, Suppl. Fig. 3 to 6). When the redox partner for AcCYP51 activity was AfCPR1 the rate of
formation of product was greater by a factor of 10 compared to using the HsCPR redox partner
2
-1
affording approximately 7.6 ± 0.05 min . Consequently, we used the AfCPR1 redox partner to
determine structure-activity.
After incubation in the usual way, no metabolism occurred with cycloartenol, whereas
obtusifoliol was fully metabolized by AcCYP51 under the same conditions as evidenced in GC-
8
,14
MS analysis showing the ∆ –product in 95% yield (Suppl. Fig. 4). Because the rate of product
formation against the AfCPR1 partner was much higher than when using the HsCPR redox
partner, we could now detect some small conversion of cycloeucalenol. This was evident in two
-1
ways; in catalytic assay to establish competence afforded a rate of 0.04 ± 0.01 min and in GC-
+
8
MS analysis showing a new cyclosteroid product (M 410, ~1% yield,) that contained the ∆ -
structure (Suppl. Fig. 4, structure 24 in Table 1). While doubtful the new metabolite converts to
5
,7
8
∆
-sterol under physiological conditions, it does shows mechanistically that the ∆ -bond is not
8
essential for CYP51 activity. In contrast, a ∆ -bond in the CYP51 substrate is required in plant
and animal sterol biosynthesis [41-43]. Except for cycloeucalenol as substrate, there was general
agreement between affinity and catalytic competence determined by the sterol difference spectra
1
7

ACCEPTED MANUSCRIPT
and binding constant (K ) and efficiency for substrate to be converted to 14-desmethyl product
d
(
kcat). Those substrates having a 9,19-cyclopropyl group, typical of intermediates in the plant
sterol biosynthesis pathway, poorly bind to the enzyme (cycloeucalenol) or not at all
cycloartenol and 24(28)-methylene cycloartanol). Structural isomers of the cyclopropyl sterol
(
7
8
that possess a ∆ -bond do not bind while those that possess a ∆ -bond bind well with obtusifoliol
Suppl. Fig 3- 10) the apparent favored substrate for AcCYP51. While not readily evident from
(
the affinity constant (K ), but apparent from the rate data (k ) through comparison of pairs of
d
cat
2
4(28)
substrates that differ in side chain construction for a ∆
–group of lanosterol (Suppl. Fig. 3 -
3
3) versus eburicol (Suppl. Fig. 3 - 32), obtusifoliol (Suppl. Fig. 3- 10) versus 31-norlanosterol
(Suppl. Fig. 3- 37), it appears that AcCYP51 prefers a substrate that possesses the 24(28)-bond
(Table 2) consistent with its phylogenetic association to land plant enzymes [6] and provides
rationale for the biosynthetic ordering of intermediates.
Thus, in good agreement with results of the titration experiments and catalytic parameters of the
AcCYP51 the enzyme clearly prefers obtusifoliol (Table 2) yielding an optimal rate for C14-
-1
demethylation of 7.6 min . The value is close to the V
reported for kinetoplastid protozoan
max
and land plant CYP51and is approximately 6 fold lower than those for the fungal and human
isoforms [6]. On the basis of the previously determined catalytic competence of cloned AcSMTs
-
1
(
referenced in Table 2), it has been shown cycloartenol [k 1.5 min /K 44 µM] is the favored
cat m
substrate for 24-SMT and the enzyme generated product is 24(28)-methylene cycloartanol while
-1
2
4(28)-methylene lophenol [k 0.8 min / K 25 µM] is the favored substrate for 28-SMT which
cat
m
2
5(27)
24(28)
converts to multiple 24-ethyl sterol ∆
- and ∆
- products [33]. Comparative analysis of
the sterol C14-demethylase and C24-methylase specificity reveal neither of the AcSMT
recognizes obtusifoliol as substrate and that AcCYP51 fails to productively accept either of the
AcSMT favored substrate.
3
.4. Identification of a new sterol biosynthesis in protozoa; evidence for separate intermediates
to C - and C -sterols.
28
29
There is no clear information about the C -sterol synthesis pathway. In the first round of
2
9
trophozoite sterol analyses, we identified 15 neutral sterols (other than the novel aromatic
sterols) from the MgCl treated cultures (compounds 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 17, 18
2
and 20, Suppl. Fig. 2 and 3) and from logarithmic phase cultures 4 more sterols were detected
1
8

ACCEPTED MANUSCRIPT
(Suppl. Fig 1 16, 19, 21 and 27 and illustrated in Suppl. Fig. 3) while additional sterols were
obtained from treated cultures corresponding to many of the compounds previously isolated from
Acanthamoeba polyphaga, including cycloartenol, 24(28)-methylene cycloartanol, obtusifoliol,
2
4β-ethyl cholesterol and 24β-methyl cholesterol [28, 29]. Our chemical identification of Ac
cycloartenol by GC-MS against a standard available to us and of parkeol synthesized from
cycloartenol by acid-induced rearrangement of the cyclopropane ring to the 9(11)-structure [44]
agrees with the careful biochemical and enzymological studies of Rohmer showing as well that
cycloartenol is the sole product of 2,3-oxidosqualene cyclization to protosterol in A. polyphaga
and 9(11)-metabolites occur as byproducts in trace amounts [28, 29]. Our observations combined
with that of Rohmer for cycloartenol rather than lanosterol as the true protosterol for
Acanthamoeba species is supported by Ac genome mining for sterolic enzymes where a single
2
,3-oxidosqualene to protosterol synthase (cyclase) is detected corresponding to the cycloartenol
synthase (CAS1). Recent phylogenetic studies have shown that CAS1 can be distinguished from
the gene for lanosterol synthase (LAS1) through a signature peptide sequence with a conserved
residue at amino acid position-442 (Ac numbering system) for isoleucine rather than valine
characteristic of the lanosterol synthase (LAS1), as shown in supplemental Figure 7 [13, 45 and
references cited therein]. Testing effects of substitution of the conserved residues in and around
position 442 (Ac numbering) on CAS1 and LAS1 have led to the notion that high conservation of
the amino acid composition in these regions represents an important structural basis for
evolutionary conservation of protosterol formation [13], and indicate that cycloartenol evolved
before lanosterol.
For elucidation of new biochemical transformations and biosynthetic pathways mass spectra of
sterols identified in GC-MS analysis are extremely powerful tools in providing information about
multiple structural features of the molecule. In many cases authentic compounds are recorded in
the recently released NIST library of chemical standards. A molecular ion is normally observed
in the mass spectrum of free sterol which is reason we have studied this sterol form in GC-MS
analysis. It is preferable to consider fragmentation patterns and subtleties in ion intensity
associated with the ring and side chain in formulating sterol structure [46]. For those that rely on
the NIST library to assign structure to sterols caution should be exercised since partial
information is obtained from observation of molecular ion and base peak of target sterol as
match indicators and thus can lead to erroneous structure assignments for several key compounds
1
9

ACCEPTED MANUSCRIPT
as reported in reference 34. Importantly, many reference sterols that occur naturally are not
entered into the NIST library. For example, the aromatic sterols identified in this study, e.g.,
+
amebasterol-1 of M 394 amu, is matched to 9-methyl anthrasteroid (anthraergostatraenol) in the
NIST library with 81.1% match of 86.7 % probability. This seemingly excellent match in the
database generates an incorrect structure for the Ac compound while the correct structure has yet
to be deposited into the NIST library. Indeed, Rohmer proved through NMR studies, and
confirmed here, the structure of this Ac metabolite as the phenanthrene skeleton harboring an
aromatic B-ring with the C19 methyl rearranged to the C6-position [31]. Another relevant
example for potential inaccuracy in structure determination as it relates to Ac sterol biosynthesis
5
,7,22
pathways comes from the supposed mass spectrum match of ergosterol which has the ∆
-
2
5(27)
24(28)
24(25)
triene system. Its ∆
-ergostene isomer, like that of its ∆
- and ∆
-ergostene isomers,
have identical mass spectra and one of more of these isomers can co-elute with ergosterol in GC
unless appropriate chromatographic conditions are met [19, 46]. Referencing the NIST library is
8
7
further limited in the identification of the set of B-ring structural isomers of ∆ versus ∆ -
5
,7(8)
8,14(15)
5,8(9)
6,8(14)
ergostenes, for the set of conjugated B/C-ring dienes of ∆
isomers and for the C24-alkyl epimers (for example) poriferasterol (24β-ethyl) and stigmasterol
24α-ethyl) [46-49]; in the usual practice, identification of the individual isomers is confirmed
, ∆
, ∆
, ∆
-ergostene
(
through a comparison of chromatographic and spectral constants of the unknown as the free
alcohol or as a C3-derivatized compound to assist in chromatographic resolution against
equivalent data obtained on authentic standards or for determination of stereochemistry at C24
through NMR of the pure compound or as necessary to synthesize the applicable standard [48].
Previously isolated sterols from Acanthamoeba are variably positioned as intermediates in
biosynthetic schemes for ergosterol and 7-dehydroporiferasterol synthesis, including a
5
hypothetical terminal step whereby ∆ - C -monoene sterols (24-ethyl cholesterol), formed from
2
9
5
,7
5,22
25(27)
∆
-sterol, convert to final ∆ -diene sterol, as can occur in land plants. Curiously, the ∆
-
C -sterols that we observed in trace amounts in A. castellanii were detected also in trace
29
amounts in A. polyphaga and A. culbertsoni by research groups with expertise in natural product
characterizations [28, 50]. However, the earlier investigators failed to consider the biosynthetic
2
5(27)
importance of ∆
-sterols to ergosterol and 7-dehydroporiferasterol synthesis, relegating them
to by-products in the sterol metabolome. The accumulation of ergsost-5-enol and poriferast-5-
enol [structures 4 and 14 in Suppl. Fig. 3] which arise from the salt treatments is problematic and
2
0

ACCEPTED MANUSCRIPT
5
,7
may not relate to their biosynthetic intermediate role in ∆ -sterol formation of ergosterol and 7-
dehydroporiferasterol as proposed [28]. Rather, these sterols may be considered metabolically
7
inert end products formed along with brassicasterol and poriferasterol from a ∆ -reductase of
broad substrate specificity during encystment and not from trophozoite growth (Fig. 6). As our
7
studies suggest, the ∆ -reductase appears only to be active during the encystment process.
2
Incubation of [methyl- H ]methionine with trophozoites revealed growth arrested cultures
3
possessed three major isotopically labeled sterols corresponding to ergosterol, poriferasterol and
+
7
-dehydroporiferasterol; for ergosterol the mass spectrum showed the dideuterated species (M
+
3
96 → M 398) while the mass spectrum of 7- dehydroporiferasterol showed the pentadeuterated
+
+
species (M 410 → M 415) and the mass spectrum of poriferasterol showed the
+
+
pentadeteuterated species (M 412 → M 417) (Figure 2, Panels E and F). The fragment
containing the high end molecular cluster at m/z 396 for ergosterol and m/z 410 for 7-
dehydroporiferasterol derived from the methyl cation on SAM was shifted by 2 Da or by 5 Da,
respectively, in the mass spectra of the labeled sterol (Suppl. Fig. 8) These labeling results show
2
4(28)
a mixed biosynthetic pathway in which ergosterol is formed by an ∆
- sterol methylation
2
pathway similar to land plants which incorporate 2 D- H -SAM atoms at C while the 7-
3
28
25(27)
dehydroporiferasterol and poriferasterol sterol C -methylation pathways are similar to the ∆
29
2
–
C -sterol methylation pathway in green algae which incorporate 5-D H -SAM atoms into the
29 3
2
C -sterol [19, 45]. In contrast, land plant C -sterol biosynthesis 4D H -SAM atoms are
29
29
3
2
4(28)
incorporated into the 24-ethyl group consistent with the involvement of a ∆
-C --sterol
29
24(25)
intermediate [51]. The enzyme-catalyzed conversion of ∆
-sterol substrate to C - and C -
28 29
sterol products were considerably altered from the sterol profile of control. As shown (Figure 2,
Panel B), the composition of the sterol mixtures obtained from cultures grown on normal and
deuterium-labeled methionine showed that the proportion of C -sterol was markedly increased
2
8
2
while the C sterols decreased in the cultures grown in the presence of [methyl- H ]methionine.
2
9
3
These labeling experiments reveal the operation of a pronounced deuterium isotope effect and
indicates that the proton loss from a C intermediate (Figure 5, Panel A) is the rate-limiting step
2
8
in formation of C -sterol products. Thus, in the presence of substrate 28-SMT affording C -
2
9
29
sterols can in some circumstances significantly alter the course and equilibrium of metabolic
sequences affecting the balance of C to C -sterols during growth and differentiation. For C -
2
8
29
29
2
5(27)
sterols there is switch to an alternate route utilizing ∆
-intermediates in contrast to the
2
1

ACCEPTED MANUSCRIPT
2
4(28)
previously reported route of ∆
-intermediates [28]. It should be remembered the cell pellet
used for our sterol analysis was from a culture composed of two populations of cells—
5
,7
5
trophozoites that synthesize ∆ -sterols and encysted cells that contain ∆ - sterols. Given the
differentiation processes discussed in section 3.1, it is more likely the poriferasterol is derived
from the 7-dehydroporiferasterol associated with trophozoite to encysted cell formation. It is also
notable the phytosterols synthesized in land plants possess side chain C24/methyl/ethyl
1
structures that are α-oriented while our HNMR analysis of the A. castellanii ergosterol and 7-
dehydroporiferasterol indicated the C24-methyl/ethyl structures are β-oriented similar to the side
chain structures of C -and C - sterols in the green alga Chlamydomona reinhardtii [19].
2
8
29
Conventional co-metabolite ordering in Ac sterol biosynthesis suggests the amoeba operates a
land plant phytosterol biosynthesis pathway via the cycloartenol to 24(28)-
2
4(28)
methylenecycloartanol ∆
-pathway to C - and C -sterol final products. However, through a
28 29
comparison of the Ac genomic database with land plant (Arabidopsis) genomic database (and for
aromatase the human genome database) together we our chemical analysis of trophozoites and
encysted cells, we reconstructed the cycloartenol to 6-methyl aromatic sterol biosynthetic
2
5(27)
pathways, taking into account phylogenetic differences in the sterol methylation of ∆
–
2
4(28)
5,7
5
(green algae) and ∆
-olefin (land plant) routes and in the formation of ∆ - sterols versus ∆ -
sterols (Fig. 6). The reconstruction of the A. castellanii sterol pathway underscores the
metabolic divergence of amoeba and recurrent evolution. The results show: (i) completion to a
5
∆
-monoene sterol atypical of green algae, (ii) selective sterol methylation for the 24β-
orientation typical of green algae (iii) in vitro AcSMT assays show obtusifoliol is not a substrate
2
5(27)
and that ∆
–sterol is a product of the enzyme catalyzed reaction consistent with ordering of
2
intermediates based on biosynthetic reasonableness, and (iv) H-methinone fed to Ac show the
2
4(28)
25(27)
C - and C - sterol products are formed by separate ∆
– C24-methylene and ∆
-24-ethyl sterols formed enzymatically
are redundant not incorporated into 7-dehydroporiferaterol under physiological conditions.
-olefin
28
29
2
4(28)
C24-ethyl intermediates, respectively, and that any ∆
3
.5. Dependence of inhibitor effect on the physiological state of the amoeba cells
The primary mechanism of the azole and steroidal inhibitor effect- inhibition of ergosterol
biosynthesis in amoeba cells- has as a prerequisite an active cellular metabolism for the
biosynthetic renewal of ergosterol with proliferation and growth. It is understandable that
2
2

ACCEPTED MANUSCRIPT
dormant cysts could only be affected at high concentrations of these drugs (>100 µM), if at all.
However, unclear is the extent to which the encysted cell can alter its sterol profile in response to
an ergosterol biosynthesis inhibitor. The aza analog of the C25-carbocation intermediate of the
C24-methylation reaction, a steroidal inhibitor, against Ac24SMT and Ac28-SMT and the tight
binding inhibitor voriconazole against AcCYP51 were utilized to determine the relative
importance of active site interactions of select sterol biosynthesis enzymes responsive to these
drugs in vitro as well as in cell-based studies
The effect of azole and 25-azacycloartanol (Fig. 7, Panel A) on rapidly proliferating trophozoites
was much the same—both compounds prevented amoeba growth at low inhibitor concentrations
of IC values 390 nM and 25 nM, respectively and MAC values between 1-3 µM (Fig. 7, Panel
5
0
B). Sterol analysis of mostly encysted cells revealed mark changes to the C - and C - sterol
2
8
29
balance associated with encystment (Suppl. Fig. 2, Panel C and Suppl. Fig. 9) and the total sterol
content was less than in trophozoite by approximately 50% on per cell or pellet basis. Although
steroidal inhibitor treatment resulted in reduced C -sterol levels, showing disruption in substrate
2
9
pools, there was not much accumulation of intermediates cycloartenol or obtusifoliol, suggesting
a down-regulation of de novo sterol synthesis as trophozoite converts to encysted cell as reported
elsewhere [35]. In neither treatment was there significant accumulation of 6-methyl aromatic
5
,7
sterol. However, after the treated cells were incubated for another 8 days all the ∆ -sterol had
converted to 6-methyl aromatic sterol accompanied by a ten- fold decline in fresh weight of the
pellet. When the concentration of inhibitor is increased 10 to 50 fold, i.e., increasing stress, the
resulting 48 cell pellet is composed exclusively of dead cells and the sterol profile consists
mostly of 6-methyl aromatic sterol.
To determine whether FUdR, a DNA synthesis inhibitor [52] that should prevent ergosterol
biosynthesis globally, had an effect on differentiation, spontaneous encystment was evaluated
after the cultures containing inhibitor reached stationary phase of control. The resulting cells had
5
a population of cysts equal to the parent trophozoite inoculum of approximately 2-3 x10
cells/ml. Effectively all of these encysted cells (>95%) were alive according to trypan blue
5
staining. The FUdR treated cells showed a significant increase in ∆ -sterol of brassicasterol and
poriferasterol (40 % total sterol) which associates with true cyst formation (Suppl. Fig. 10). In
contrast, for treatments with voriconazole or 25-azacyloartanol the number of cells at 48 h was
2
3

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4
reduced by a factor of 10 to 2-3 x 10 cells/ml, most were dead by trypan blue staining (Fig. 7,
Panel B) and they (encysted cells) possessed mostly ergosterol and 7-dehydroporiferasterol
(Suppl. Fig. 9) with no accompanying 6-methyl aromatic sterol.
Time-killing experiments were carried out by the addition of inhibitor at approximate MAC
levels to the culture medium for 48 incubations. Figure 7 (Panel C) shows that medium
5
inoculated with a population of 1 x 10 cells/ml trophozoites (90%) affords continued
trophozoite proliferation, without significant encystment (data not shown), to a stationary phase
6
population of 1 x 10 cell/ml. Alternatively, in the presence of the test inhibitors, at about the
MAC, the trophozoite population drops to zero. This stressing of amoeba stimulates trophozoite
to encyst, which as inhibitor pressure is maintained yields a decrease in the total number of
4
2
encysted cells from approximate 3 x 10 encysted cells/ml at 12 h to an approximate 5 x10 to 5
3
x10 cells/ml at 48 h growth. Trypan blue staining of the encysted cells revealed two dominant
populations characterized by those that are viable or non-viable and dead. When the 48 h treated
pellets were transferred to fresh medium and incubated for a week, 25-azacycloartanol
treatments showed no growth while from the voriconazole treatment a few thousand trophozoites
were evident, suggesting the predominant population of encysted cells viewed as dead by trypan
blue staining were indeed dead in terms of excystment.
Because the clumped cells were in much greater density in treated cells than in control, it was
straightforward to examine them by scanning electron microscopy (SEM) and transmission
election microscopy (TEM). Thus, SEM and TEM images of 25-azacycloartanol-generated cysts
(VOR looked much the same) from the 48 h harvest were virtually the same as those of control
cysts originating in T-flasks showing one or more flaccid cells clumped together with rounded
cells (cf. Figure 2, Panel C). Fine structure of the treated cells also showed a deformation in cell
wall associated with an aberrant thickening or partially formed ectocyst and endocyst, and the
cell interior looked severely damaged with (i) formation of blebby structures in the cell
membrane, (ii) broken membrane structures and (iii) intense vacuolization and loss of
mitochondria typically associated with autophagy-like structures in the center of the cell that
contained undigested organelle.
To determine whether inhibitors designed to block two essential enzymes in the post-squalene
segment of the ergosterol biosynthesis pathway are more potent in combination than when added
2
4

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to the medium singly, we monitored the effects of combining subinhibitory concentrations of one
drug against varied concentrations of the other drug on trophozoite multiplication. Complete
eradication of the amoeba parasite is obtained when using the drugs in combination at
concentrations as low as equal to their IC values which in our studies for voriconazole was 140
5
0
nM ± 10 nM compared to our previously reported 390 nm [32] and for 25-azacycloartanol is 25
nM ± 2 nM (Fig. 7, Panel D). The number of dead cells at 48 h in the combination drug
experiment of 25 nM 25-azacycloartanol (25 nM) and voriconazole (100 nM) was similar to the
number of dead cells when the drugs were administered individually to the medium (Fig. 7,
Panel B), and no trophozoites were detected after 5 days following resuspension of treated pellet
into fresh medium. These results suggest that combined treatment of two or more rationally
designed inhibitors at sub-MAC concentration can successfully interfere with sterol biosynthesis
to prevent leakiness at the target enzyme and in so doing limits production of the downstream
C - and C -sterols, supporting a role for AcCYP51 and AcSMTs in the control of metabolic flux
28
29
through distinct intermediates.
.6 Excystment in response to inhibitor treatment.
3
In a separate experiment the anti-amoeba effect of the drugs on excystment was determined with
cysts derived from the SDS-treated process. Using MAC concentrations of the ergosterol
biosynthesis inhibitors, all the resting cysts convert to trophozoites by 5 days, indicating that
excystment is insensitive to these treatments. In similar fashion, the FUdR treated resting cells
easily excysted into trophozoites by 3 days. During these experiments, increase numbers of
empty cyst walls were evident in cell mixture, consistent with excystment.
3
.7. Enzymatic basis of action and selectivity.
All the available experimental evidence indicates that AcCYP51 and 24-/28-AcSMT are the
primary target of the medical azole and steroidal N-inhibitor, respectively, since we observed
small accumulations of obtusifoliol and cycloartenol in the presence of voriconazole and
2
4(R,S),25-epiminolanosterol [32, 33]. The target enzymes were characterized from cloned genes
introduced into E. coli and solubilized for activity assay. In our earlier study, we observed that
voriconazole is a specific and reversible inhibitor of AcCYP51 exhibiting an IC of 390 nM and
5
0
its MAC is approximately 3 µM [32, 33]. 25-Azacycloartanol tested as a true substrate analog
2
5

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inhibitor is approximately as effective against 24-AcSMT and 28-AcSMT as voriconazole
against AcCYP51. The inhibitor against trophozoite proliferation generates an IC of 25 nM ±
5
0
0
.25 nM and MAC of 1 µM ± 0.05 µM (data not shown) and against 24-SMT incubated with
cycloartenol as substrate or 28-SMT incubated with 24(28)-methylene lophenol as substrate
yields IC values of 15 nm (K = 13 nM) and 22 nM (K = 8 nM) (Suppl. Fig. 11). The measured
5
0
i
i
inhibition of the AcCYP51 and SMT activities is quantitatively sufficient to account for the
inhibition of ergosterol and 7-dehydroporifersterol biosynthesis in amoeba.
3
.8. Effect of Acanthamoeba sterols on growth of GL7
To assess the structural specificity for sterols as membrane inserts directly, we used the yeast
GL7 auxotrophic for sterol. Close interactions between sterol supplementation and the growth
response was detected. Ergosterol supplementation to GL7 afforded growth arrest in 72 h
8
affording approximate 1 x 10 cells/ml, typical of wild-type yeast, while cultures without added
ergosterol yielded a no growth-response, as reported previously (Fig. 8) [8, 39, 53]. Ergosterol
was recovered in unchanged form from the cells at approximately 20 fg/cell. Microscopic
examination showed the GL7 yeast at growth arrest revealed mostly single cells or those that
were undergoing budding. Alternate sterol supplements fed to GL7, 7-dehydroporiferasterol,
8
brassicasterol, poriferasterol reached a stationary phase of 1 x 10 cells/ml similar to the
ergosterol fed cells and each of the sterols was recovered from cells in unchanged form at 20
fg/cell. Alternatively, cells fed the 6-methyl aromatic sterol supplement failed to proliferate by
7
2 and microscopically many cells were clumped or dead by trypan blue staining typical of yeast
cultured on a steroidal inhibitor [53]. Past interpretation using the GL7 system showing a no
growth response following administration of nutritionally adequate amounts of sterol is the
supplement failed to adequately fit the membrane structure [7, 8].
2
6

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DISCUSSION
In spite of the therapeutic importance for an exacting sterol composition in parasitic protozoa
27], relatively little is known about the regulation and function of phytosterols in amoeba. This
[
lack of information on metabolic controls is a serious impediment to the development of
strategies for targeted inhibition of phyla-specific enzymes, such as the sterol C24-methylase,
that can occur in the protozoan pathogen while absent from the human host. Previous functional
and phylogenetic analysis of amoeba steroidogenesis has left the role and origins of C - and C -
2
8
29
sterols somewhat opaque. In particular, while it seems clear the Acanthamoeba can synthesize
ergosterol and 7-dehydroporiferasterol, it has been assumed there is one set of terminal
5
,7
compounds – ∆ -sterols - that act unaltered as membrane inserts and for the sterol biosynthetic
genes to be descended from plants. However, the possibility for evolutionary accommodation of
5
5,7
stage-specific differences in ∆ -, ∆ - and 6-methyl aromatic compounds in Acanthamoeba was
indicated by GC-MS analysis of mixed populations of trophozoites and encysted cells, although
5
5,7
it was ignored, leading to the view that ∆ - sterols are the precursor of ∆ -sterols, that the C24-
2
4(28)
methylation ∆
-route typical of land plants yield intermediates for the synthesis of C - and
28
C -sterols and for the sterolic enzymes to lack substrate specificity affording criss-crossing
29
5
,7
routes of major and minor biosynthesis pathways to final ∆ -sterol products [2, 29, 54].
Subsequent co-metabolite and phylogenetic analysis based on an erroneous characterization of
many of the reported amoeba sterols, incorrect bioinformatic analyses for identity of the 2,3-
oxidosqualene cyclase as lanosterol synthase and failure to report a SMT2-type isoform required
in the synthesis of 24-ethyl sterols [34] developed an enigmatic history for Acanthamoeba
ergosterol biosynthesis pathway as originating with the fungi.
Here and in our previous paper [33] we provide evidence for the first time that indicates the
sterol biosynthetic enzymes in Acanthamoeba were composed of catalytically constrained
participants of strict substrate specificity, for sterol methylases to be responsible for generating
2
4(28)
25(27)
distinct ∆
- and ∆
-olefin intermediates that convert to functional C - and C -sterol
28 29
products of varied nucleus structures and that the conventional sterols typical of protozoa
ergosterol and 7-dehydroporiferaterol can be replaced with the uncommon brassicasterol-
poriferasterol and amebasterol-1 and amebasterol-2 steroidal pairs during the encystment phase
of the amoeba life history. Because methylation patterns in large part determine product
2
7

ACCEPTED MANUSCRIPT
outcome, subtle alterations in methyltransferase substrate selectivity have a profound impact on
the balance of C - and C -sterols, which was noted following inhibitor treatment. The step-wise
2
8
29
alteration in the sterol compositions observed during a normal excystment-encystment cycle
5
5,7
yielding ∆ -sterols and 6-methyl aromatic sterols from a ∆ -sterol parent compound is not
consistent with the limited sterol diversity in protozoa, rather it supports a simple model of
5
,7
cryptic unikont evolution not necessarily observed in the bikont amoeba Naegleria wherein a ∆
5
–
sterol biosynthetic renewal is crucial to trophozoite proliferation, ∆ -sterol synthesis is essential
for the production of a resting cyst capable of excystment and 6-methyl aromatic sterols, derived
5
,7
from ∆ -sterols in encysted cells considered non-viable for excystment, promote cell lysis and
death as outlined in the proposed model of Acanthamoeba sterol biosynthesis and function
shown in Figure 9.
Comparing the ontogenetically regulated sterol compositions in trophozoites and encysted cells
and catalytic analysis of relevant chokepoint enzymes AcCYP51 and AcSMT is phylogenetically
interesting yet connects to the functional importance of sterol products through modified
structural features that occur in the stage-specific furnished molecule. Thus, proliferating
5,7
trophozoites synthesize C - and C -∆ -sterol products typical of kinetoplastids, such as
2
8
29
Trypanosma cruzi, and the green algae synthesize ergosterol and 7-dehydroporiferasterol [19, 20,
7] but these organisms synthesize the C - and C -sterols through different biosynthesis routes.
5
2
8
29
It appears AcCYP51 possesses substrate preference and catalytic competence more like that of
plant, and hence protozoa, than of fungal or human CYP51 [6]. The substrate specificity of
AcCYP51 places C14-demethylation after the first sterol methylation step (Fig. 6). However,
2
4(25)
24(28)
while T. cruzi has a single SMT1 which is ∆
- and ∆
-substrate bifunctional capable of
2
4(28)
generating a single ∆
bifunctional capable of generating a single ∆
SMTs to carry out these sterol methylations at C24 with the second methylation step yielding the
-Z ethylidene C – product [55-57] and green alga SMT is substrate
2
9
2
5(27)
-C -sterol product [8], A. castellanii has two
29
2
4β-stereochemistry. As we discovered in this investigation through isotopically labeling studies
2
5(27)
to amoeba, the AcSMT2 (or 28-SMT) can, unlike the T. cruzi SMT1, synthesize the C -∆
-
2
9
olefin outcome and the resulting intermediate is precursor to the 7-dehydroporiferasterol and
2
4(28)
poriferasterol. Fungi, on the other hand, synthesize a single SMT1 operating the ∆
-route
specific for zymosterol and ergosterol while land plants possess two SMTs and both operate the
2
4(28)
∆
–route such that separate intermediates are involved in the formation of C α-campesterol
28
2
8

ACCEPTED MANUSCRIPT
2
5(27)
and C α-sitosterol, respectively [20]. The lineage-specific recurrence of an ancient ∆
-sterol
2
9
metabolic pathway affording primitive 24β-ethyl sterols in the unikonts suggest divergence with
the ancestral SMT occurred independently multiple times during the course of eukaryote
evolution and within the protozoa product-specific families of SMTs evolved unique to the
Acanthamoeba. The notion of divergent evolution rather than convergent evolution for the
amoeba SMTs is supported by the conservation of sequence similarity and gene structure among
the diverse SMTs isolated throughout the eukaryotic domain to date [8, 59, 60].
The results of studies carried out so far on stage-specific sterol profiling in Acanthamoeba and
for GL7 growth responses to sterol supplementation lead to the important conclusion that the
membranes of trophozoites and resting cysts have certain structural requirements for sterol that
are remarkably similar to yeast. They are clearly associated with the three-dimensional character
of the molecule since C - and/or C -sterols are consistently found in the cells. In the
28
29
5
,7
trophozoite and resting cyst, the sterols possess a double bond in ring B of ∆ -bond
5
5
(
trophozoite) or ∆ -bond (resting cyst). The terminal formation of ∆ - sterols could represent a
fine tuning in nucleus planarity but the structural change does not qualify for a significant effect
5
,7
5
on membrane fluidity. We surmise the conserved metabolism of ∆ -sterols to form a ∆ -sterol
5
relates to its homoallylic effect on the C-O bond at C3. For the ∆ -sterol, donation of electrons
through an inductive effect, analogously to the well-studied 3,5-cyclosteroid rearrangement,
should strengthen the H-O bond and therefore weaken somewhat and fine-tune any hydrogen
bonding of the sterol to phospholipid in the lipid leaflet of the resting cyst. It should be
remembered that steric interference from C4-methyl groups on the hydrogen bonding ability of
C3- group can weaken sterol-lipid interactions and therefore is reason for removal of C4 to
generate an acceptable sterol membrane insert.
5
,7
In depending upon ∆ -sterols to act as the encysted cell sterols, it is important not to overlook
5
,7
these cells are on a trajectory for lysis and death, which associate with rapid turnover of the ∆ -
sterol, now as intermediates, to a 6-methyl aromatic sterol; cell death from these sterols is
observed in GL7 growth experiments as well. The B-ring aromatic sterols effectively replace
5
,7
∆
-sterols as the membrane component. It appears the architectural unsuitability of the
phenanthrene sterols stems mainly from their diminished hydrogen-bonding strength of the C3-
group resulting from the altered electronegativity of the polar group affected by the double bond
2
9