Download Autophagy is involved in starvation response and cell

Microbiology (2010), 156, 665–677
DOI 10.1099/mic.0.033944-0
Autophagy is involved in starvation response and
cell death in Blastocystis
Jing Yin, Angeline J. J. Ye and Kevin S. W. Tan
Correspondence
Laboratory of Molecular and Cellular Parasitology, Department of Microbiology, Yong Loo Lin
School of Medicine, National University of Singapore, 5 Science Drive 2, Singapore
Kevin S. W. Tan
[email protected]
Received 20 August 2009
Revised
20 October 2009
Accepted 9 November 2009
Previous studies have demonstrated that colony forms of Blastocystis undergo cell death with
numerous membrane-bound vesicles containing organelles located within the central vacuole,
resembling morphological features of autophagy. In this study, we investigated whether
Blastocystis underwent autophagy upon amino acid starvation and rapamycin treatment.
Concurrently, we provide new insight into a possible function of the central vacuole. The use of
the autophagy marker monodansylcadaverine, and the autophagy inhibitors 3-methyladenine and
wortmannin, showed the existence of autophagy in amino-acid-starved and rapamycin-treated
Blastocystis. Confocal microscopy and transmission electron microscopy studies also showed
morphological changes that were suggestive of autophagy. The unusually large size of the
autophagic compartments within the parasite central vacuole was found to be unique in
Blastocystis. In addition, autophagy was found to be triggered when cells were exposed to the
cytotoxic antibody mAb 1D5, and autophagy was intensified in the presence of the caspase
inhibitor zVAD.fmk. Taken together, our results suggest that the core machinery for autophagy is
conserved in Blastocystis, and that it plays an important role in the starvation response and cell
death of the parasite.
INTRODUCTION
Autophagy, in particular macroautophagy, is the major
mechanism used by eukaryotic cells to degrade long-lived
proteins, and it is perhaps the only known pathway for
degrading organelles (Levine & Klionsky, 2004). It is
believed to be a conserved process in all eukaryotic cells.
During autophagy, a double-membrane structure, known
as the phagophore, forms and expands to sequester a
portion of cytoplasm in the form of an autophagosome.
The autophagosome fuses with a lytic compartment,
the engulfed materials are degraded, and the resulting
macromolecules are recycled (Klionsky & Emr, 2000;
Levine & Klionsky, 2004). In yeast, autophagy is induced
under adverse conditions, such as nutrient deficiency, to
degrade proteins that are not needed, and recycle the
amino acids for survival (Takeshige et al., 1992; Yorimitsu
& Klionsky, 2005). In mammals, autophagy has been
implicated in development, differentiation and disease
Abbreviations: LC3, light chain 3; 3-MA, 3-methyladenine; MAP LC3b,
microtubule-associated protein light chain 3b; MDC, monodansylcadaverine; MOMP, mitochondrial outer-membrane permeability; PI3K,
phosphatidylinositol 3-kinase; TEM, transmission electron microscopy;
TOR, target of rapamycin; zVAD.fmk, N-benzyloxycarbonyl-Val-AlaAsp(O-Me)-fluoromethylketone.
A Western blot of LC3-like antigen in Blastocytis, MDC staining of
Blastocystis cells treated with rapamycin, and a table of Atg homologues
in the Blastocystis EST database are available with the online version of
this paper.
033944 G 2010 SGM
processes (Kuma et al., 2004; Levine & Klionsky, 2004;
Shintani & Klionsky, 2004).
In contrast to the extensive studies of autophagy in yeast
and higher eukaryotes, the mechanism and significance of
autophagy in the more primitive protozoan parasites is far
less well understood. Recent reports have indicated an
increasing interest in this field. Autophagy has been
observed in dying Tetrahymena thermophila cells following
staurosporine treatment (Christensen et al., 1998). In
Leishmania donovani, treatment with antimicrobial peptides induces cell death via autophagy (Bera et al., 2003).
Autophagy has been found to be essential for differentiation and virulence of Leishmania major, and cysteine
proteases have been found to be necessary for the process
(Besteiro et al., 2006; Williams et al., 2006). In
Trypanosoma, candidate autophagy genes have been
identified through a bioinformatic search (Herman et al.,
2006), and some components of the Atg8 conjugation
system have been verified to function in differentiation of
Trypanosoma cruzi (Alvarez et al., 2008a, b). Recently,
autophagy has been found to play an important role in the
proliferation and differentiation of the enteric protozoan
parasite Entamoeba (Picazarri et al., 2008).
Blastocystis is a protozoan parasite that inhabits the large
intestines of humans and many other animals (Tan, 2004,
2008). The parasite is prevalent in developing countries,
and it has been associated with intestinal diseases. It is a
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 15 Jun 2017 21:55:23
Printed in Great Britain
665
J. Yin, A. J. J. Ye and K. S. W. Tan
polymorphic organism with four commonly described
forms: vacuolar, granular, amoeboid and cystic. The
vacuolar form is commonly seen in the stools of patients
with intestinal disorders, and this form is the predominant
cell type seen in axenized in vitro cultures. In this form of
Blastocystis, a thin rim of cytoplasm surrounds a characteristic large central vacuole that takes up to 90 % of the
cell volume (Tan, 2008). However, little is known about the
function of the enigmatic central vacuole (Tan, 2004).
Although there has been no report of autophagy in
Blastocystis, previous studies have shown unexplained
morphological features suggestive of autophagy. For
example, ageing Blastocystis cells, grown as colonies, have
been found to have cytoplasmic vacuolation or membranebound vesicles containing organelles within the central
vacuole (Tan et al., 2001). These observations have been
seen mostly in cells located in the centre of the colony. As
these cells have limited access to nutrients compared with
cells at the periphery, it is likely that the autophagic
machinery has been triggered in response to nutrient
deprivation (Tan et al., 2001; Tan & Nasirudeen, 2005).
Given the importance of autophagy in the wide array of
eukaryotic cellular processes, and its possible role in host–
pathogen interactions (Alvarez et al., 2008b; Colombo,
2007), we aimed to verify the existence of autophagy in
Blastocystis, and elucidate the role of autophagy in the
survival and proliferation of this parasite. Furthermore, the
study of Blastocystis autophagy may shed light on the
function of the unique central vacuole of this parasite.
METHODS
Culture of organism. Blastocystis subtype 7 (formerly known as
Blastocystis hominis isolate B) was obtained from a local patient, and
maintained axenically in Iscove’s modified Dulbecco’s medium
(IMDM) containing 10 % inactivated horse serum, as described
(Ho et al., 1993). Cells were grown anaerobically using an AnaeroJar
(Oxoid) at 37 uC. Organisms were subcultured at 3–4 day intervals.
Autophagy induction. Blastocystis cells were subjected to various
treatments for the assessment of autophagy. To test for the presence
of autophagy in Blastocystis colonies, cells were grown in soft agar, as
previously described (Tan et al., 1996b), with minor modifications.
Approximately 16104 cells taken from day 4 exponential-phase
cultures were suspended in 1 ml IMDM, and this was added to 20 ml
of a mixture containing 0.36 % Bacto agar and 0.1 % sodium
thioglycollate in IMDM supplemented with 10 % horse serum, at
40 uC. The agar was poured into a 100615 mm Petri dish
(Nunclon). After gentle swirling to ensure good separation of cells,
the Petri dish was kept at 220 uC for 10 min to allow the agar to set,
and then it was incubated in an anaerobic jar at 37 uC for 10 days. For
amino acid starvation, 16107 Blastocystis cells were washed three
times in PBS, and incubated in 5 ml Hanks’ balanced salts solution
(HBSS). The tubes were then incubated anaerobically at 37 uC for
different time intervals (1, 2 or 3 h). Rapamycin treatment was
carried out by incubating 16107 Blastocystis cells with 100, 500 or
1000 nM rapamycin (Sigma) in 5 ml IMDM supplemented with 10 %
horse serum, and cultures were incubated anaerobically at 37 uC for
3 h. A DMSO control was also included. The cytotoxic monoclonal
antibody mAb 1D5 [produced previously in our laboratory (Tan et al.,
666
1996a)], against Blastocystis, was also tested to see whether an
autophagy response was triggered. Cells were pretreated with
50 mM N-benzyloxycarbonyl-Val-Ala-Asp(O-Me)-fluoromethylketone (zVAD.fmk; Sigma) and/or 10 mM cyclosporin A (Sigma) for
30 min, and they were then exposed to mAb 1D5 or a non-specific
IgM (derived from the snake Bungarus multicinctus; Nasirudeen &
Tan, 2005), and incubated anaerobically for 24 h at 37 uC.
Monodansylcadaverine staining. Following various treatments for
autophagy induction, cells in liquid culture were spun down, washed
once in PBS, and resuspended in 500 ml PBS. Then a 10 ml volume of
5 mM monodansylcadaverine (MDC; Fluka) in PBS was added to the
suspension, and it was incubated at 37 uC for 15 min. After
incubation, the cells were washed three times in PBS, and mounted
on a glass slide under a coverslip. Cells were then visualized with a
fluorescence microscope (Olympus BX60) using an excitation filter of
360 nm and an emission filter of 525 nm. The images were captured
by a CCD camera (Olympus DP70). The percentage of MDC-positive
cells was determined by scoring 500 cells per group.
To stain colony cultures of Blastocystis, colonies embedded in the agar
were removed by a sterile inoculating loop, and stained with 0.05 mM
MDC in PBS for 30 min. Then the colonies were washed twice in
1 ml PBS, with a 5 min interval between each wash. The colonies were
then placed on a slide, and pressed gently with a coverslip. The
colonies were viewed under a fluorescence microscope to examine the
uptake of the stain by the cells at the centre and at the periphery of the
colony.
Effect of the autophagy inhibitors 3-methyladenine and
wortmannin. To test the effect of the autophagy inhibitors 3-
methyladenine (3-MA) and wortmannin on Blastocystis, cells were
pretreated with 20 mM 3-MA (Sigma) or 50 mM wortmannin, and
they were incubated for 3 h prior to amino acid starvation, rapamycin
treatment or exposure to mAb 1D5, as described above.
Western blotting. Blastocystis cells were incubated in IMDM
medium or HBSS for 1–3 h. Cells were lysed in buffer (25 mM
Tris, pH 7.5, 150 mM NaCl, 0.1 % Triton X-100, 1 mM DTT, 1 mM
EDTA, 1 mM EGTA, 10 mM NaF, 20 mM b-glycerophosphate,
1 mM Na3VO4, and Roche complete protease inhibitor), and the
soluble fraction was obtained by centrifugation (14 500 g, 10 min) at
4 uC. The total protein concentration was determined by the Bradford
assay. The total protein (50 mg) was electrophoresed on a 12 % SDSPAGE gel, and transferred to a PVDF membrane (GE Healthcare) by
semi-dry blotting (Bio-Rad). The membrane was then blocked with
5 % skim milk in PBS-T (16 PBS with 0.1 % Tween-20), incubated
with rabbit anti-human microtubule-associated protein light chain 3b
(MAP LC3b) polyclonal antibody (dilution 1 : 500; Santa Cruz
Biotechnology), washed in PBS-T and incubated in a 1 : 10000
dilution of horseradish-peroxidase-conjugated goat anti-rabbit
immunoglobulin (Santa Cruz Biotechnology). The proteins were
detected by chemiluminescence (ECL plus; GE Healthcare) and
exposed to an X-ray film (Kodak).
BLAST
searches
of
ATG
orthologues
in
Blastocystis.
Bioinformatic analysis was carried out to search for orthologues of
AuTophaGy-related (ATG) genes in Blastocystis. To date, 32 ATG
genes have been identified in Saccharomyces cerevisiae and other fungi
(Kanki et al., 2009; Okamoto et al., 2009). Protein sequences of the
yeast ATGs were retrieved from Swiss-Prot (www.ebi.ac.uk/uniprot/),
and used as queries to perform a BLAST (Basic Local Alignment Search
Tool; http://blast.ncbi.nlm.nih.gov/Blast.cgi) search against the
Blastocystis EST database (http://tbestdb.bcm.umontreal.ca/searches/
organism.php?orgID=BH) stored locally using StandAlone BLAST
version 2.2.18.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 15 Jun 2017 21:55:23
Microbiology 156
Autophagy in Blastocystis
Confocal microscopy examination of MDC and Lysotracker
Red costaining. Following various treatments, the cell pellet was
stained with 0.05 mM MDC for 15 min at 37 uC, and then with
50 nM Lysotracker Red DND-99 (Molecular Probes) for a further
30 min at 37 uC. After incubation, the cells were washed three times
in PBS, and mounted on glass slides under coverslips. Fluorescent
images were obtained by using a confocal microscope (Olympus
Fluoview FV500). MDC was excited at 405 nm, and the emission was
collected at 505–525 nm. Lysotracker Red was excited at 543 nm, and
the emission was collected at 584–654 nm.
Transmission electron microscopy. The ultrastructural features of
Blastocystis cells under various treatments were examined using
transmission electron microscopy (TEM). Briefly, cells were fixed
with 2 % glutaraldehyde and 2 % paraformaldehyde in PBS for 3 h at
4 uC. The cells were then pelleted at 1000 g for 5 min, and washed
twice in PBS, and twice in deionized water. After each washing step,
cells were spun at 1500 g, and the supernatant was discarded. Cells
were then post-fixed for 2 h with 1 % osmium tetroxide containing
1 % potassium ferrocyanide at room temperature, followed by
dehydration with a graded ethanol series, and infiltration and
embedding with LR-White resin (London Resin Company).
Ultrathin sections were stained with uranyl acetate and lead citrate,
and examined using an EM208S transmission electron microscope
(Philips).
Statistical analysis. Experiments were repeated at least twice.
Quantitative data were statistically evaluated using Student’s t test,
and differences were considered significant at the level of P,0.05.
Values are given as means±SD.
RESULTS
MDC accumulates in the central region of the
Blastocystis colony
MDC is an autofluorescent compound that has been
reported to specifically label autophagic vacuoles in in vivo
and in vitro conditions because of its ability to act as a
lysosomotropic agent and as a solvent polarity probe
(Biederbick et al., 1995; Munafo & Colombo, 2001;
Niemann et al., 2000). Since the ultrastructural features
of Blastocystis colonies have implied the occurrence of
autophagy, MDC labelling was performed in Blastocystis
colonies. After 10 days of incubation, discrete buffcoloured opaque colonies were observed macroscopically
in agar inoculated with Blastocystis cells. Using an inverted
microscope, distinct biconvex-disc-shaped colonies
embedded in the soft agar were observed. Individual
colonies without compromised integrity were stained with
MDC to examine the uptake of the stain by the cells at the
centre and the periphery of the colony (Fig. 1). While a
certain level of background staining was seen in all cells,
true MDC-positive staining was identified by the prominent fluorescence intensity. It was observed that cells
located in the centre of the colony showed a significant
proportion of MDC-positive cells, whereas cells located at
the periphery of the colony showed virtually no MDCpositive staining. The data suggest that autophagy may
indeed be triggered in cells located at the centre of the
colony.
MDC incorporation increases with amino acid
starvation and rapamycin treatment in
Blastocystis
Because it has been shown in other eukaryotic cells that
autophagy is often rapidly upregulated when cells are
under nutritional stress (Levine & Klionsky, 2004; Munafo
& Colombo, 2001; Takeshige et al., 1992), Blastocystis was
deprived of amino acids to further study and ascertain the
autophagy phenomenon in this organism.
Blastocystis cells were incubated in IMDM medium
(control) or in HBSS for 1–3 h. After treatment, cells were
stained with MDC (Fig. 2), and the MDC-positive cells
were scored. The percentage of MDC-positive cells
increased from 6.7±0.9 % in cells incubated in IMDM
medium to 19.7±2.2 % (P,0.05) after 1 h amino acid
starvation. There was also a time-dependent increase of
MDC-positive cells as the starvation progressed to 2 h
(28.6±0.2 %; P,0.05) and 3 h (38.0±1.4 %; P,0.05)
periods. Hence, these results suggested that autophagy was
rapidly triggered in Blastocystis cells in response to amino
acid starvation.
Fig. 1. MDC staining of colony forms of Blastocystis. Blastocystis cells were grown as colonies in soft agar. On day 10, intact
colonies embedded in the agar were removed by using an inoculating loop, stained with 0.05 mM MDC, and viewed under a
fluorescence microscope. Arrows indicate representative MDC-positive cells.
http://mic.sgmjournals.org
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 15 Jun 2017 21:55:23
667
J. Yin, A. J. J. Ye and K. S. W. Tan
Fig. 2. MDC staining of Blastocystis cells
deprived of amino acids for 1, 2 or 3 h. Cells
were washed three times in PBS, and incubated in HBSS anaerobically at 37 6C for
different times (1, 2 or 3 h). The control (ctrl)
was performed by incubating cells in IMDM
supplemented with 10 % inactivated horse
serum. 3-MA and wortmannin (WT) treatments
were performed by incubating cells with
20 mM 3-MA or 50 mM WT for 3 h prior to
amino acid starvation for a further 3 h.
Following the different incubation periods,
cells were stained with 0.05 mM MDC, and
viewed under a fluorescence microscope.
Representative fluorescent images of MDC
staining are shown; arrows point to cells
stained positive for MDC.
Target of rapamycin (TOR) is a conserved Ser/Thr kinase,
and is a central controller of cell growth. It is a negative
regulator of autophagy, and inhibition of TOR leads to
induction of autophagy (Meijer & Codogno, 2004).
Nutrient starvation or absence of growth factors can
inhibit TOR, and trigger autophagy in yeast and mammalian cells (Lum et al., 2005; Wullschleger et al., 2006). It
is very likely that the TOR signalling pathway also exists
and functions in Blastocystis, and that the accumulation of
MDC staining observed in serum-starved Blastocystis cells
was due to the inactivation of TOR. To verify the existence
of the TOR signalling network in Blastocystis, we
investigated whether rapamycin, the prototypical inhibitor
of TOR, could induce autophagy in Blastocystis.
Blastocystis cells were treated with 100, 500 or 1000 nM
rapamycin for a period of 3 h. MDC staining was visually
scored using fluorescence microscopy (Fig. 3). Similar to
amino acid starvation, punctuate intensive staining by
MDC was observed in rapamycin-treated cells. As compared with the DMSO control (8.0±1.0 % MDC-positive
cells), cells that had been incubated with 1000 nM
rapamycin had a significantly higher percentage of MDCpositive cells (45.5±12.4 %; P,0.05). Rapamycin at a
concentration of 100 or 500 nM did not induce a
significant change in the number of MDC-positive cells:
12.0±4.5 % and 14.2±5.6 %, respectively. These results
indicate that rapamycin induced autophagy in Blastocystis,
and thus the TOR signalling pathway is likely to be
conserved in Blastocystis. Trypanosome TOR has been
reported to have an IC50 of 152 nM for rapamycin after
treatment for 72 h (Barquilla et al., 2008). The fact that
Blastocystis was insensitive to rapamycin concentrations
below 1000 nM may be due to the brief incubation period
of 3 h. Supplementary Fig. S2 shows that after incubation
with rapamycin for 72 h, Blastocystis exhibited a dosedependent increase in the percentage of MDC-positive cells
at rapamycin concentrations of 100, 500 and 1000 nM.
Fig. 3. MDC staining of Blastocystis cells
treated with rapamycin for 3 h. Cells were
incubated with 100, 500 or 1000 nM rapamycin in IMDM supplemented with 10 % horse
serum. The cultures were incubated anaerobically at 37 6C for 3 h. The control (ctrl) was
contained DMSO. 3-MA and wortmannin (WT)
treatments were performed by incubating cells
with 20 mM 3-MA or 50 mM WT for 3 h prior
to rapamycin treatment for 3 h. After the
incubation period, cells were stained with
0.05 mM MDC, and viewed under a fluorescence microscope. Representative fluorescent images of MDC are shown; arrows
point to cells stained positive for MDC.
668
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 15 Jun 2017 21:55:23
Microbiology 156
Autophagy in Blastocystis
Inhibitory effects of 3-MA and wortmannin on
MDC incorporation
To evaluate whether the incorporation of MDC was indeed
dependent on autophagy, cells were pretreated with 3-MA
or wortmannin, which are inhibitors of autophagic
sequestration. 3-MA and wortmannin are phosphatidylinositol 3-kinase (PI3K) inhibitors, and they inhibit both
class I and class III PI3K. Class I PI3K generates products
that inhibit autophagic sequestration, while class III PI3K
products stimulate autophagic sequestration downstream
of class I enzymes; therefore, the overall effect of 3-MA and
wortmannin is to block autophagy (Blommaart et al., 1997;
Petiot et al., 2000). The addition of 3-MA to 3 h aminoacid-starved cells decreased the percentage of MDCpositive cells from 38.0±1.4 % to 9.5±0.6 % (P,0.05
versus control consisting of Blastocystis cells in IMDM
medium), and the addition of wortmannin decreased the
percentage to 5.5±3.1 % (Fig. 2). A similar inhibitory
effect was seen in rapamycin treatment, as the MDC uptake
of cells treated with 1000 nM rapamycin dropped from
45.5±12.4 % to 8.14±0.6 % and 8.7±1.6 % upon the
addition of 3-MA and wortmannin, respectively (Fig. 3).
Therefore, in the presence of 3-MA or wortmannin,
there was an inhibitory effect on the incorporation of
MDC in both amino-acid-starved and rapamycin-treated
Blastocystis cells.
Homology search of ATG genes, and detection of
LC3-like antigen in Blastocystis
searches for Atg orthologues in the Blastocystis EST
database generally resulted in very low scores
(Supplementary Table S1), which may be due to the
incompleteness of the database. Microtubule-associated
protein light chain 3 (LC3) or Atg 8 homologues are widely
used to monitor autophagy in various organisms including
protozoan parasites (Alvarez et al., 2008a; Klionsky et al.,
2008). However, no significant match was found in a BLAST
search of the Blastocystis EST database (Table S1). Using a
rabbit polyclonal antibody raised against amino acids 1–50
mapping at the N terminus of human MAP LC3b, we
identified a LC3-like antigen in Blastocystis cell lysates
(Supplementary Fig. S1). The size of the LC3-like antigen
in Blastocystis was 20 kDa, which is slightly higher than
that of the mammalian homologue of LC3-I (16 kDa)
(Mizushima, 2004). Interestingly, the LC3-like antigen of
Blastocystis underwent a time-dependent degradation when
cells were under amino acid starvation conditions for
different periods of time.
BLAST
MDC staining colocalizes with Lysotracker Red
staining in the central vacuole
It was observed that most of the MDC staining accumulated in the central vacuole. To elucidate the function of
the Blastocystis central vacuole in autophagy, morphological changes of amino-acid-starved and rapamycinhttp://mic.sgmjournals.org
treated cells were assessed via confocal microscopy after costaining with MDC and Lysotracker Red. Lysotracker Red
is a fluorophore that selectively accumulates in acidic
compartments, such as lysosomes, whereas MDC was first
described as a specific marker of autophagic vacuoles
(Biederbick et al., 1995). By double staining, the dynamics
of the autophagy process were anticipated because early
autophagosomes should be stained with MDC only, and
after their fusion with lysosomes co-localization can be
observed. However, Fig. 4 shows that MDC and
Lysotracker Red staining almost always co-localized.
MDC or Lysotracker Red single-labelled controls were
checked to ensure no cross-talk or bleed-through of the
two fluorescent dyes (results not shown). The results were
not surprising, since the specificity of MDC staining has
been cautioned by other workers, and it has been suggested
that MDC is similar to other acidotropic dyes (eg,
Lysotracker Red), and that it may preferentially label later
stages in the degradation process of autophagy. Early
autophagosomes may not be readily labelled by MDC
because they are not acidic (Bampton et al., 2005; Klionsky
et al., 2008; Mizushima, 2004). Nonetheless, these results
suggest that, in Blastocystis, MDC and Lysotracker Red
label the same autophagic compartments of acidic pH,
although the detailed nature of the structure awaits further
investigation.
Examinations of the confocal microscopy images revealed
very unique MDC staining patterns in Blastocystis. Most of
the extensive staining was seen inside the central vacuole as
either individual circular specks or irregular-shaped
objects, which were likely to be clusters of several specks.
The size of MDC-labelled individual specks was 0.5–1 mm,
which is comparable to the size of autophagosomes in yeast
(0.3–0.9 mm) (Yorimitsu & Klionsky, 2005). However, the
size of the irregular-shaped objects was 2–8 mm, and this
has not be found in yeast or mammalian cells, except for
the 5–10 mm autophagosomes containing bacteria or
protozoan parasites (Andrade et al., 2006; Nakagawa et
al., 2004). Interestingly, in another enteric protozoan
parasite, Entamoeba invadens, the sizes of autophagosome-like structures were very similar to those we found in
Blastocystis (Picazarri et al., 2008). In that study, the size of
Atg8-associated structures varied from ,1 mm to .4 mm,
and the appearance of the large-sized structures coincided
with the initiation of encystation process.
Ultrastructural features of Blastocystis under
nutrient stress
Amino acid-starved or rapamycin-treated Blastocystis cells
displayed ultrastructural features resembling those of
autophagy (Fig. 5). As compared with the untreated
control (Fig. 5a, b), most of the treated cells showed
cytoplasmic vacuoles with membranous inclusions, and
numerous vacuoles were observed to be accumulating
within the central vacuole. The content of the autophagiclike vacuoles included intact mitochondria, membrane
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 15 Jun 2017 21:55:23
669
J. Yin, A. J. J. Ye and K. S. W. Tan
Fig. 4. Confocal microscopy images of Blastocystis incubated under amino acid starvation condition for 3 h, or in 1000 nM
rapamycin for 3 h. Cells were incubated with 1000 nM rapamycin in IMDM supplemented with 10 % horse serum, or were
incubated in HBSS (amino acid starved), anaerobically at 37 6C for 3 h. The control contained DMSO. Following the
incubation, cells were stained with 0.05 mM MDC for 15 min at 37 6C, and then with 50 nM Lysotracker Red DND-99 for a
further 30 min at 37 6C, and then they were analysed by using a confocal microscope. DIC, differential interference contrast.
Bars, 10 mm.
whirls and amorphous materials. The presence of vacuoles
containing membranous inclusions or intact organelles is
highly suggestive of autophagy. The size of most of the
autophagic vacuoles observed in TEM was 0.5–1 mm, while
some were 2 mm (Fig. 5l), 4 mm (Fig. 6a) and 6 mm (Fig. 5i
arrow) in size. This is consistent with the size of MDClabelled structures observed by confocal microscopy. Less
frequently, it was observed that cytoplasmic contents were
invaginating through vesicle-like or thin-tube-like membrane structures into the central vacuole (Fig. 6).
Interestingly, there were vesicles budding at the end of
the filament-like structures. We hypothesize that the
autophagic vacuoles were formed in different ways:
membrane expansion, and sequestration of cytoplasmic
material in the cytoplasm (Fig. 5j); extensive vacuolation in
cytoplasmic regions near to the central vacuole, and
formation of vesicles that invaginate into the central
vacuole (Fig. 6a–d); formation of a thin-tube-like structure
that extrudes into the central vacuole, and budding-off
vesicles at the end of the tubular invagination into the
670
central vacuole (Fig. 6e–g). We posit that the central
vacuole is a reservoir for most autophagic vacuoles.
Lysosomes are likely to be translocated from the cytoplasm
into the central vacuole in the meantime, and fuse with
newly formed autophagic vacuoles (Fig. 5e, f). Our
observation that some autophagic vacuoles displayed
ultrastructural evidence for double membranes characteristic of such structures, surrounded by the central vacuole
membrane, seems to support this model (Fig. 5g, i).
mAb 1D5 induces autophagic features in
Blastocystis
Previous studies have demonstrated that the cytotoxic
monoclonal antibody mAb 1D5 induces apoptotic features
in Blastocystis; these features included externalization of
plasma membrane phosphatidylserine, mitochondrial
outer-membrane permeabilization, and DNA fragmentation (Nasirudeen & Tan, 2004; Nasirudeen et al., 2001).
While apoptotic features can be inhibited by the caspase
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 15 Jun 2017 21:55:23
Microbiology 156
Autophagy in Blastocystis
Fig. 5. Ultrastructural observations of autophagic vacuoles deposited in the central vacuole of Blastocystis. Untreated controls
(a, b) displayed normal cell morphology. The central vacuole contains flocculent contents. Nu, nucleus; M, mitochondrion; G,
Golgi apparatus; L, lipid inclusion; CV, central vacuole. Amino-acid-starved cells (c–j) and rapamycin-treated cells (k–n)
showed numerous autophagic-like vacuoles within the central vacuole. (f) A mitochondrion appears to be in the process of
fusion with a lysosome [enlarged from the upper box in (e)]. (g) Several double-membrane vesicles with more disintegrated
contents [enlarged from the lower box in (e)]; the inset shows a region with three layers of membranes. (i) A vesicle containing
two mitochondria and cytoplasm (arrow), a vacuole containing membranous whirls (dashed arrow and inset), and another
double-membrane vacuole (arrowhead) [enlarged from (h)]. (J) A double membrane formed in the cytoplasm (arrow) and a
cytoplamic vacuole (dashed arrow) [enlarged from (h)]. (l) A vacuole with a mitochondrion and some cytoplasmic material
[enlarged from (k)]. (n) Two vesicles with an inner limiting membrane (arrows), and another vesicle containing enclosed
membrane sacs and a lipid granule (dashed arrow) [enlarged from the box in (m)]. Bars, 2 mm (a, b, h), 1 mm (c, d, e, i, j, k, m),
0.5 mm (f, g, l, n).
inhibitor zVAD.fmk, and the mitochondrial outer-membrane permeability (MOMP) inhibitor cyclosporin A, the
cells cannot be rescued from death (Nasirudeen & Tan,
2005). It has been suggested that mAb 1D5 can elicit a
programmed-cell-death response in Blastocystis that is
independent of caspases, mitochondria, or both, probably
through an alternative pathway other than apoptosis
(Nasirudeen & Tan, 2005; Tan & Nasirudeen, 2005).
Since autophagic cell death has often been triggered as an
alternative cell death pathway when apoptosis is blocked
http://mic.sgmjournals.org
(Gozuacik & Kimchi, 2004), it was of interest to investigate
whether mAb 1D5 could induce autophagic cell death in
Blastocystis.
In order to evaluate the possibility of autophagic cell death
in Blastocystis treated with mAb 1D5, alone and in the
presence of zVAD.fmk and/or cyclosporin A, cells were
labelled with MDC. The cells in each treatment were
visually scored for MDC-positive staining by fluorescence
microscopy (Fig. 7). Cells treated with growth medium or a
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 15 Jun 2017 21:55:23
671
J. Yin, A. J. J. Ye and K. S. W. Tan
non-specific IgM monoclonal antibody, alone or in the
presence of zVAD.fmk and/or cyclosporin A, showed a
small percentage of MDC-positive cells (,15 %). In
contrast, mAb-1D5-treated cells had a higher percentage
(18.9 %) of MDC-positive cells compared with the control.
In the presence of pan-caspase inhibitor zVAD.fmk, 37.9 %
of mAb-1D5-treated cells showed MDC positivity.
However, when cells were exposed to mAb 1D5 in the
presence of cyclosporin A, the percentage of MDC-positive
cells decreased to 11.6 %. mAb-1D5-treated Blastocystis
pre-exposed to zVAD.fmk and cyclosporin A showed
10.8 % MDC-positive cells. The autophagy inhibitor 3-MA
was also found to have an inhibitory effect on the
incorporation of MDC with mAb 1D5 treatment. These
results indicate that the phenomenon of autophagy was
triggered by mAb 1D5 treatment, and was intensified in the
presence of zVAD.fmk. In addition, the mAb-1D5-elicited
autophagy might be associated with the induction of
MOMP.
DISCUSSION
In this study, we present what is believed to be the first
description of autophagy in the protozoan parasite
Blastocystis through MDC labelling and ultrastructural
characterization. Strong and punctate MDC staining was
observed in cells located in the centre of a Blastocystis
colony. Within a colony, the cells in the centre have less
access to nutrients and, hence, they are most likely to
undergo starvation, as compared with the cells at the
periphery of the colony. In this study, cells grown in liquid
culture and deprived of amino acids also had a high
percentage of MDC-positive staining. In addition, timedependent increase of MDC staining was observed during
the first 3 h of amino acid starvation. These results suggest
a strong correlation between MDC staining and the
nutritional state of the cell. Since MDC has been reported
to stain autophagic vacuoles, and autophagy is rapidly
upregulated in response to nutrient-deficient conditions in
other organisms (Takeshige et al., 1992; Yorimitsu &
Klionsky, 2005), the intensive MDC staining should be a
good reflection of the autophagic activity in Blastocystis,
and it was used in further investigation. The results also
demonstrated that autophagy occurs naturally when cells
have limited access to nutrients, and that it can be rapidly
induced by removing nutrients from the culture medium.
Fig. 6. Ultrastructural observations of cytoplasmic invagination
into the central vacuole when cells were deprived of amino acids
(a, b and e), or treated with rapamycin (c, d, f and g). (a–d)
Invagination of membrane-bound vesicles into the central vacuole.
Multiple vacuolations were seen in the cytoplasmic region near the
central vacuole, and some vesicles appeared to be in the process
of invaginating into the central vacuole. (e–g) Invagination of
membrane-bound filaments into the central vacuole, with vesicles
located at the tip of the filament (arrows). Bars, 2 mm (a, c, f, g),
1 mm (b, d, e).
672
Results of the current study suggest that some of the
autophagy machinery is conserved in Blastocystis. TOR is a
conserved Ser/Thr kinase, and a negative regulator of
autophagy. Rapamycin induces autophagy through inhibition of TOR (Meijer & Codogno, 2004). It was found that,
in Blastocystis, treatment with rapamycin elicited a similar
response to that of amino acid starvation. Rapamycin
treatment was effective in intensifying MDC-labelled
vesicles; hence, Blastocystis is likely to have a homologue
of TOR, and the TOR signalling pathway should also exist
and function in Blastocystis. The PI3K inhibitors 3-MA and
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 15 Jun 2017 21:55:23
Microbiology 156
Autophagy in Blastocystis
wortmannin inhibited the incorporation of MDC effectively. Both class I and class III PI3K have been reported to
regulate autophagy in yeast and mammalian cells. Class I
PI3K generates products to inhibit autophagic sequestration, while class III PI3K products stimulate autophagic
sequestration downstream of class I enzymes, so the overall
effect of 3-MA and wortmannin is to block autophagy
(Blommaart et al., 1997; Petiot et al., 2000). Inhibition of
MDC incorporation by 3-MA and wortmannin suggests
that a similar regulation pathway exists in Blastocystis, and
also confirms the autophagic nature of MDC-labelled
vesicles. An LC3-like antigen was found via cross-reactivity
of anti-human LC3 antibody. In other studies with
mammalian cells, upon initiation of macroautophagy, the
cytosolic form LC3-I is conjugated with phosphatidylethanolamine, and becomes the membrane-bound form LC3II, which is specifically located on isolation membranes and
autophagosomes (Klionsky et al., 2008; Mizushima, 2004).
Immunoblotting of LC3 upon autophagy induction will
thus show the conversion of LC3-I to LC3-II. However, the
current study detected only one reactive band using antihuman antibody. The possibility of the antibody binding to
an unrelated protein that shares the same epitopes as LC3
cannot be excluded, and the identity of this LC3-like
antigen needs to be confirmed in a future study.
Nonetheless, the degradation of LC3-like antigen may
reflect the non-selective degradation of cellular proteins
during the autophagy process.
Morphological examination by confocal microscopy and
TEM revealed several unique features of autophagy in
Blastocystis. Most of the extensive MDC staining was
observed inside the central vacuole, and this suggests a role
of this organelle in the autophagy process. MDC-stained
structures appeared as either individual circular vesicles of
size 0.5–1 mm, or big clusters of several specks of size 2–
8 mm. Similar large-sized structures in autophagy have
been described in only one other intestinal protozoan
parasite, E. invadens, and their appearance coincided with
the initiation of the encystation process (Picazarri et al.,
2008). Since starvation is known to induce encystation in
protozoan parasites such as Giardia lamblia and E.
invadens (Avron et al., 1986; Lujan et al., 1996), it is
possible that starvation triggered some Blastocystis cells to
transit into granular or cyst forms, and that autophagy may
promote the morphological changes through massive
clusters of autophagic vacuoles. TEM is the most reliable
method for monitoring autophagy (Mizushima, 2004).
Highly polymorphic autophagic vacuoles were observed in
amino-acid-starved and rapamycin-treated cells. Based on
these randomly observed profiles, we postulate that the
process of autophagy in Blastocystis starts from cytoplasmic
membrane expansion to enclose organelles and other
materials, and the formed autophagic vacuoles with double
membranes are deposited into the central vacuole via
invagination, so the resulting autophagic vacuoles exhibit
three layers of membranes. The other scenario is the direct
transfer of cytoplasmic components through vesicle
http://mic.sgmjournals.org
invagination or tubular invagination, and subsequent
budding of vesicles into the central vacuole, leading to
the formation of autophagic vacuoles with one layer of
membrane. Thus, the outer membrane of all the autophagic vacuoles found in the central vacuole is likely to be
formed from the membrane of central vacuole via the
invagination process. These single-membrane-bound
structures eventually fuse with invaginated lysosomes.
Because TEM images represent very thin sections of the
sample, there is a possibility that the autophagic vacuoles
observed in the central vacuoles are connected to the
peripheral cytoplasm. Regardless of the origin, most
autophagic vacuoles appeared to be accumulated in the
central vacuole. The central vacuole is the largest organelle
in Blastocystis vacuolar forms, and takes up approximately
90 % of the volume of the cell. It has been speculated to be
an important organelle of this organism; however, the
function of central vacuole is largely unknown (Tan, 2008).
Through amino acid starvation and rapamycin treatment,
the current study has suggested a role of the central vacuole
in autophagy. Other studies using biochemical staining for
carbohydrates or lipids have shown that some cells
accumulate these two substances in the central vacuole,
while some cells do not show any positive reactions to the
stainings (Yoshikawa et al., 1995a, b). It is likely that the
lipids and carbohydrates are actually released from
degraded autophagic vacuoles, as autophagy serves to
maintain homeostasis in healthy cells. The current study
could also shed light on numerous unexplained observations with the central vacuole in earlier studies.
Cytoplasmic inclusions within the central vacuole have
been reported by others (Dunn et al., 1989; Pakandl, 1999),
and they may be due to baseline autophagy in the parasites.
The cytoplasmic projections into the central vacuole have
been shown to increase in number during unfavourable
culture conditions, such as in the presence of high
concentrations of antibiotics (Boreham & Stenzel, 1993).
It is possible that the cells upregulated autophagy in
response to environmental stress.
It has been reported that mAb-1D5-treated Blastocytis preexposed to zVAD.fmk and/or cyclosporin A is not rescued
from cell death (Nasirudeen & Tan, 2004, 2005). In the
presence of either inhibitor, there seemed to be a
compensatory mechanism, as the cells were able to trigger
the mitochondrial-dependent death pathway in the absence
of caspase-like activity, and vice versa. However, even
though both caspase-like activity and MOMP were
inhibited, and DNA fragmentation was abolished completely, the cells continued to die. Therefore, besides
apoptosis, other cell death pathways might exist in
Blastocystis, and be triggered upon mAb 1D5 induction.
MDC-labelled vesicles were observed in mAb-1D5-treated
Blastocystis, and 18.9 % of cells showed MDC-positive
staining, suggesting that autophagy was triggered by mAb
1D5. The interplay between apoptosis and autophagy is
complex, and not fully understood. Similar stimuli can
induce either apoptosis or autophagy concomitantly,
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 15 Jun 2017 21:55:23
673
J. Yin, A. J. J. Ye and K. S. W. Tan
674
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 15 Jun 2017 21:55:23
Microbiology 156
Autophagy in Blastocystis
Fig. 7. Exposure to mAb 1D5 increased MDC-positive staining in Blastocystis. Cells were pretreated with 50 mM zVAD.fmk
and/or 10 mM cyclosporin A (CA) for 30 min, and then incubated with mAb 1D5 or a non-specific IgM, anaerobically for 24 h at
37 6C. 3-MA treatment was performed by incubating cells with 20 mM 3-MA for 3 h prior to exposure to mAb 1D5. Following
the different treatments, cells were stained with 0.05 mM MDC, and examined under a fluorescence microscope. (a)
Fluorescent images of MDC staining of healthy and treated Blastocytis cells; bar 10 mm. (b) MDC staining of healthy and mAb1D5-treated Blastocystis cells in the presence of 3-MA; bar 10 mm. (c) Bar chart comparing MDC-positive Blastocystis cells
under different conditions. Five hundred cells were counted in each treatment. Values are means (±SD) of two separate
experiments, *P,0.05.
sequentially, or in a mutually exclusive manner (Maiuri et
al., 2007). It appeared that mAb 1D5 could trigger both
apoptosis and autophagy, although apoptosis was still the
major mode of cell death. However, when Blastocystis was
pre-exposed to zVAD.fmk before incubation with mAb
1D5, the percentage of MDC-positive cells increased to
37.9 %, indicating an upregulation of autophagy when
caspase-like activity was blocked. This is consistent with
findings in many metazoan cell lines that were sensitized to
autophagic cell death in the absence of caspase activation
(Vandenabeele et al., 2006; Yu et al., 2004). The current
study also demonstrated the importance of MOMP in
mAb-1D5-initiated autophagy, as when MOMP was
inhibited by cyclosporin A, MDC-positive staining was
abolished in mAb-1D5-treated cells, even with pretreatment of zVAD.fmk. Mitochondrial damage has been
reported to be a signal for autophagy in mammalian cells.
Overexpression of mitochondrial calpain 10 has been
found to cause mitochondrial swelling and increased
autophagy, which was blocked by cyclosporin A
(Arrington et al., 2006). It is likely that autophagy was
triggered in Blastocystis via mAb-1D5-mediated mitochondrial dysfunction.
the central vacuole of this organism plays an important
role as a repository of autophagic vacuoles in the
autophagy process.
ACKNOWLEDGEMENTS
This work was supported by a generous grant (R-182–000–090–305)
from the Biomedical Research Council, Singapore. We are grateful to
Mrs Josephine Howe for assistance with TEM.
REFERENCES
Alvarez, V. E., Kosec, G., Sant’Anna, C., Turk, V., Cazzulo, J. J. & Turk, B.
(2008a). Autophagy is involved in nutritional stress response and
differentiation in Trypanosoma cruzi. J Biol Chem 283, 3454–3464.
Alvarez, V. E., Kosec, G., Sant Anna, C., Turk, V., Cazzulo, J. J. &
Turk, B. (2008b). Blocking autophagy to prevent parasite differenti-
ation: a possible new strategy for fighting parasitic infections?
Autophagy 4, 361–363.
Andrade, R. M., Wessendarp, M., Gubbels, M. J., Striepen, B. &
Subauste, C. S. (2006). CD40 induces macrophage anti-Toxoplasma
gondii activity by triggering autophagy-dependent fusion of pathogen-containing vacuoles and lysosomes. J Clin Invest 116, 2366–2377.
Arrington, D. D., Van Vleet, T. R. & Schnellmann, R. G. (2006).
In this study, autophagy was found to be involved in the
response to nutritional stress, and in cytotoxic-antibodymediated cell death. Triggering autophagy in response to
nutrient scarcity may represent an adaptive response by
generating recycled metabolic substrates to maintain
energy homeostasis (Meijer & Codogno, 2004; Tsukada &
Ohsumi, 1993). However, when the nutrient supply via
autophagy becomes ultimately depleted in prolonged
starvation, massive autophagy may contribute to cell death
(Eskelinen, 2005; Galluzzi et al., 2008). Autophagy may also
serve as an alternative pathway for cells to die when they
are stressed, such as with exposure to the cytotoxic
antibody mAb 1D5, and it may become the main pathway
of cell death when classical apoptosis is inhibited. The
cellular target of mAb 1D5 has been recently identified to
be a cell surface legumain (Wu et al., 2010), which is an
asparagine endopeptidase that has been suggested to
participate in helminth alimentary digestion of host
proteins (Oliver et al., 2006). We postulate that inhibition
of legumain activity by mAb 1D5 hinders the nutritional
uptake of Blastocystis, and thus elicits a starvation response
in the parasite.
In summary, we report the occurrence of autophagy in
Blastocystis in response to various cellular stresses, and that
http://mic.sgmjournals.org
Calpain 10: a mitochondrial calpain and its role in calcium-induced
mitochondrial dysfunction. Am J Physiol Cell Physiol 291, C1159–
C1171.
Avron, B., Stolarsky, T., Chayen, A. & Mirelman, D. (1986).
Encystation of Entamoeba invadens IP-1 is induced by lowering the
osmotic pressure and depletion of nutrients from the medium. J
Protozool 33, 522–525.
Bampton, E. T., Goemans, C. G., Niranjan, D., Mizushima, N. &
Tolkovsky, A. M. (2005). The dynamics of autophagy visualized in live
cells: from autophagosome formation to fusion with endo/lysosomes.
Autophagy 1, 23–36.
Barquilla, A., Crespo, J. L. & Navarro, M. (2008). Rapamycin inhibits
trypanosome cell growth by preventing TOR complex 2 formation.
Proc Natl Acad Sci U S A 105, 14579–14584.
Bera, A., Singh, S., Nagaraj, R. & Vaidya, T. (2003). Induction of
autophagic cell death in Leishmania donovani by antimicrobial
peptides. Mol Biochem Parasitol 127, 23–35.
Besteiro, S., Williams, R. A., Morrison, L. S., Coombs, G. H. &
Mottram, J. C. (2006). Endosome sorting and autophagy are essential
for differentiation and virulence of Leishmania major. J Biol Chem
281, 11384–11396.
Biederbick, A., Kern, H. F. & Elsasser, H. P. (1995). Monodansyl-
cadaverine (MDC) is a specific in vivo marker for autophagic
vacuoles. Eur J Cell Biol 66, 3–14.
Blommaart, E. F., Krause, U., Schellens, J. P., Vreeling-Sindelarova, H.
& Meijer, A. J. (1997). The phosphatidylinositol 3-kinase inhibitors
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 15 Jun 2017 21:55:23
675
J. Yin, A. J. J. Ye and K. S. W. Tan
wortmannin and LY294002 inhibit autophagy in isolated rat hepatocytes. Eur J Biochem 243, 240–246.
Nakagawa, I., Amano, A., Mizushima, N., Yamamoto, A., Yamaguchi, H.,
Kamimoto, T., Nara, A., Funao, J., Nakata, M. & other authors (2004).
Boreham, P. F. & Stenzel, D. J. (1993). Blastocystis in humans and
Autophagy defends cells against invading group A Streptococcus. Science
306, 1037–1040.
animals: morphology, biology, and epizootiology. Adv Parasitol 32, 1–
70.
Nasirudeen, A. M. & Tan, K. S. (2004). Caspase-3-like protease
Christensen, S. T., Chemnitz, J., Straarup, E. M., Kristiansen, K.,
Wheatley, D. N. & Rasmussen, L. (1998). Staurosporine-induced cell
influences but is not essential for DNA fragmentation in Blastocystis
undergoing apoptosis. Eur J Cell Biol 83, 477–482.
death in Tetrahymena thermophila has mixed characteristics of both
apoptotic and autophagic degeneration. Cell Biol Int 22, 591–598.
Nasirudeen, A. M. & Tan, K. S. (2005). Programmed cell death in
Colombo, M. I. (2007). Autophagy: a pathogen driven process.
Blastocystis hominis occurs independently of caspase and mitochondrial pathways. Biochimie 87, 489–497.
IUBMB Life 59, 238–242.
Nasirudeen, A. M. A., Tan, K. S. W., Singh, M. & Yap, E. H. (2001).
Dunn, L. A., Boreham, P. F. L. & Stenzel, D. J. (1989). Ultrastructural
Programmed cell death in a human intestinal parasite, Blastocystis
hominis. Parasitology 123, 235–246.
variation of Blastocystis hominis stocks in culture. Int J Parasitol 19,
43–56.
Eskelinen, E. L. (2005). Doctor Jekyll and Mister Hyde: autophagy
Niemann, A., Takatsuki, A. & Elsasser, H.-P. (2000). The lysosomo-
tropic agent monodansylcadaverine also acts as a solvent polarity
probe. J Histochem Cytochem 48, 251–258.
can promote both cell survival and cell death. Cell Death Differ 12
(Suppl. 2), 1468–1472.
Okamoto,
Galluzzi, L., Vicencio, J. M., Kepp, O., Tasdemir, E., Maiuri, M. C. &
Kroemer, G. (2008). To die or not to die: that is the autophagic
Mitochondria-anchored receptor Atg32 mediates degradation of
mitochondria via selective autophagy. Dev Cell 17, 87–97.
question. Curr Mol Med 8, 78–91.
Oliver, E. M., Skuce, P. J., McNair, C. M. & Knox, D. P. (2006).
Gozuacik, D. & Kimchi, A. (2004). Autophagy as a cell death and
Identification and characterization of an asparaginyl proteinase
(legumain) from the parasitic nematode, Haemonchus contortus.
Parasitology 133, 237–244.
tumor suppressor mechanism. Oncogene 23, 2891–2906.
Herman, M., Gillies, S., Michels, P. A. & Rigden, D. J. (2006).
Autophagy and related processes in trypanosomatids: insights from
genomic and bioinformatic analyses. Autophagy 2, 107–118.
Ho, L. C., Singh, M., Suresh, G., Ng, G. C. & Yap, E. H. (1993). Axenic
culture of Blastocystis hominis in Iscove’s modified Dulbecco’s
medium. Parasitol Res 79, 614–616.
Kanki, T., Wang, K., Cao, Y., Baba, M., Klionsky, D. J., Okamoto, K.,
Kondo-Okamoto, N. & Ohsumi, Y. (2009). Atg32 is a mitochondrial
protein that confers selectivity during mitophagy. Dev Cell 17, 98–
109.
K.,
Kondo-Okamoto,
N.
&
Ohsumi,
Y.
(2009).
Pakandl, M. (1999). Blastocystis sp. from pigs: ultrastructural changes
occurring during polyxenic cultivation in Iscove’s modified
Dulbecco’s medium. Parasitol Res 85, 743–748.
Petiot, A., Ogier-Denis, E., Blommaart, E. F. C., Meijer, A. J. &
Codogno, P. (2000). Distinct classes of phosphatidylinositol 39-
kinases are involved in signaling pathways that control macroautophagy in HT-29 cells. J Biol Chem 275, 992–998.
Picazarri, K., Nakada-Tsukui, K. & Nozaki, T. (2008). Autophagy
during proliferation and encystation in the protozoan parasite
Entamoeba invadens. Infect Immun 76, 278–288.
Klionsky, D. J. & Emr, S. D. (2000). Autophagy as a regulated pathway
of cellular degradation. Science 290, 1717–1721.
Shintani, T. & Klionsky, D. J. (2004). Autophagy in health and disease:
Klionsky, D. J., Abeliovich, H., Agostinis, P., Agrawal, D. K., Aliev, G.,
Askew, D. S., Baba, M., Baehrecke, E. H., Bahr, B. A. & other authors
(2008). Guidelines for the use and interpretation of assays for
Takeshige, K., Baba, M., Tsuboi, S., Noda, T. & Ohsumi, Y. (1992).
monitoring autophagy in higher eukaryotes. Autophagy 4, 151–175.
a double-edged sword. Science 306, 990–995.
Autophagy in yeast demonstrated with proteinase-deficient mutants
and conditions for its induction. J Cell Biol 119, 301–311.
Kuma, A., Hatano, M., Matsui, M., Yamamoto, A., Nakaya, H.,
Yoshimori, T., Ohsumi, Y., Tokuhisa, T. & Mizushima, N. (2004). The
Tan, K. S. (2004). Blastocystis in humans and animals: new insights
role of autophagy during the early neonatal starvation period. Nature
432, 1032–1036.
Tan, K. S. (2008). New insights on classification, identification, and
Levine, B. & Klionsky, D. J. (2004). Development by self-digestion:
Tan, K. S. & Nasirudeen, A. M. (2005). Protozoan programmed cell
using modern methodologies. Vet Parasitol 126, 121–144.
clinical relevance of Blastocystis spp. Clin Microbiol Rev 21, 639–665.
molecular mechanisms and biological functions of autophagy. Dev
Cell 6, 463–477.
death – insights from Blastocystis deathstyles. Trends Parasitol 21,
547–550.
Lujan, H. D., Mowatt, M. R., Byrd, L. G. & Nash, T. E. (1996).
Tan, S. W., Ho, L. C., Moe, K. T., Chen, X. Q., Ng, G. C., Yap, E. H. &
Singh, M. (1996a). Production and characterization of murine
Cholesterol starvation induces differentiation of the intestinal parasite
Giardia lamblia. Proc Natl Acad Sci U S A 93, 7628–7633.
Lum, J. J., DeBerardinis, R. J. & Thompson, C. B. (2005). Autophagy
monoclonal antibodies to Blastocystis hominis. Int J Parasitol 26,
375–381.
in metazoans: cell survival in the land of plenty. Nat Rev Mol Cell Biol
6, 439–448.
Tan, S. W., Singh, M., Thong, K. T., Ho, L. C., Moe, K. T., Chen, X. Q.,
Ng, G. C. & Yap, E. H. (1996b). Clonal growth of Blastocystis hominis
Maiuri, M. C., Zalckvar, E., Kimchi, A. & Kroemer, G. (2007). Self-
in soft agar with sodium thioglycollate. Parasitol Res 82, 737–739.
eating and self-killing: crosstalk between autophagy and apoptosis.
Nat Rev Mol Cell Biol 8, 741–752.
Tan, K. S., Howe, J., Yap, E. H. & Singh, M. (2001). Do Blastocystis
Meijer, A. J. & Codogno, P. (2004). Regulation and role of autophagy
hominis colony forms undergo programmed cell death? Parasitol Res
87, 362–367.
in mammalian cells. Int J Biochem Cell Biol 36, 2445–2462.
Tsukada, M. & Ohsumi, Y. (1993). Isolation and characterization of
Mizushima, N. (2004). Methods for monitoring autophagy. Int J
Biochem Cell Biol 36, 2491–2502.
autophagy-defective mutants of Saccharomyces cerevisiae. FEBS Lett
333, 169–174.
Munafo, D. B. & Colombo, M. I. (2001). A novel assay to study
Vandenabeele, P., Vanden Berghe, T. & Festjens, N. (2006). Caspase
autophagy: regulation of autophagosome vacuole size by amino acid
deprivation. J Cell Sci 114, 3619–3629.
inhibitors promote alternative cell death pathways. Sci STKE 2006,
pe44.
676
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 15 Jun 2017 21:55:23
Microbiology 156
Autophagy in Blastocystis
Williams, R. A., Tetley, L., Mottram, J. C. & Coombs, G. H. (2006).
Yoshikawa, H., Kuwayama, N. & Enose, Y. (1995a). Histochemical
Cysteine peptidases CPA and CPB are vital for autophagy and
differentiation in Leishmania mexicana. Mol Microbiol 61,
655–674.
detection of carbohydrates of Blastocystis hominis. J Eukaryot
Microbiol 42, 70–74.
Wu, B., Yin, J., Texier, C., Roussel, M. & Tan, K. S. (2010). Blastocystis
Yoshikawa, H., Satoh, J., Enose, Y., Keenan, T. W., Huang, C. M. &
Zierdt, C. H. (1995b). Light and electron microscopic localization of
legumain is localized on the cell surface and specific inhibition of its
activity implicates a pro-survival role for the enzyme. J Biol Chem. (in
press), doi:10.1074/jbc.M109.049064.
lipids in Blastocystis hominis. Comparative analysis of lipid composition in axenic strains of Blastocystis hominis. J Electron Microsc
(Tokyo) 44, 100–103.
Wullschleger, S., Loewith, R. & Hall, M. N. (2006). TOR Signaling in
Yu, L., Alva, A., Su, H., Dutt, P., Freundt, E., Welsh, S., Baehrecke, E. H.
& Lenardo, M. J. (2004). Regulation of an ATG7-beclin 1 program of
growth and metabolism. Cell 124, 471–484.
autophagic cell death by caspase-8. Science 304, 1500–1502.
Yorimitsu, T. & Klionsky, D. J. (2005). Autophagy: molecular
machinery for self-eating. Cell Death Differ 12 (Suppl. 2), 1542–1552.
http://mic.sgmjournals.org
Edited by: L. J. Knoll
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 15 Jun 2017 21:55:23
677