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Invited Review
Membrane trafficking and organelle biogenesis in Giardia lamblia: Use it or lose it
Carmen Faso, Adrian B. Hehl
⇑
Institute of Parasitology, University of Zurich, Winterthurerstrasse 266a, 8057 Zurich, Switzerland
article info
Article history:
Received 1 December 2010
Received in revised form 23 December 2010
Accepted 24 December 2010
Available online 4 February 2011
Keywords:
Biopolymer
Encystation
Peripheral vesicle
Mitosome
Antigenic variation
Reductive evolution
Golgi
abstract
The secretory transport capacity of Giardia trophozoites is perfectly adapted to the changing environment
in the small intestine of the host and is able to deploy essential protective surface coats as well as mol-
ecules which act on epithelia. These lumen-dwelling parasites take up nutrients by bulk endocytosis
through peripheral vesicles or by receptor-mediated transport. The environmentally-resistant cyst form
is quiescent but poised for activation following stomach passage. Its versatility and fidelity notwithstand-
ing, the giardial trafficking systems appear to be the product of a general secondary reduction process
geared towards minimization of all components and machineries identified to date. Since membrane
transport is directly linked to organelle biogenesis and maintenance, less complexity also means loss
of organelle structures and functions. A case in point is the Golgi apparatus which is missing as a
steady-state organelle system. Only a few basic Golgi functions have been experimentally demonstrated
in trophozoites undergoing encystation. Similarly, mitochondrial remnants have reached a terminally
minimized state and appear to be functionally restricted to essential iron–sulfur protein maturation pro-
cesses. Giardia’s minimized organization combined with its genetic tractability provides unique opportu-
nities to study basic principles of secretory transport in an uncluttered cellular environment. Not
surprisingly, Giardia is gaining increasing attention as a model for the investigation of gene regulation,
organelle biogenesis, and export of simple but highly protective cell wall biopolymers, a hallmark of
all perorally transmitted protozoan and metazoan parasites.
Ó2011 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction
Infection with Giardia lamblia (syn. Giardia intestinalis,Giardia
duodenalis) is one of the major causes of diarrheal disease with
an estimated 250 million clinical cases worldwide (WHO, 1997).
As an intestinal lumen-dwelling parasite which can transform into
an environmentally-resistant cyst form, Giardia interfaces with its
environments via secreted proteins and carbohydrates. The most
striking result of secretory activity is a prominent cyst wall, which
is deposited on the surface of encysting cells and whose descrip-
tion dates back to Grassi (1879). The discovery of the variant-spe-
cific surface protein (VSP) coat and antigenic variation pointed to
additional major protein trafficking pathways to the plasma mem-
brane of trophozoites. In addition to classical exocytosis of mem-
brane proteins, there is also evidence for non-conventional
secretion of metabolic enzymes and factors which modulate the re-
sponse of the gut epithelium to infection.
Endocytic uptake and transport of nutrients from the complex
environments in both the natural habitat and in cell culture is
not as well understood. In contrast to the other Diplomonads, the
Giardiinae lack a cytostome organelle with which to handle the
bulk of fluid phase transport. Instead, a unique organellar system
consisting of peripheral vesicles (PVs) which is arrayed just below
the plasma membrane appears to provide an all-in-one solution to
nutrient uptake, digestion and retrograde transport of building
blocks to the interior of the cell (Lanfredi-Rangel et al., 1998). Dur-
ing the past two decades, several research groups have investi-
gated intracellular protein trafficking in greater detail using
rigorous and systematic approaches. This work was initiated by a
report on the transport of cyst wall material (CWM) in encysta-
tion-specific vesicles in differentiating trophozoites (Reiner et al.,
1990). It became clear that despite an intracellular organization
with strongly reduced complexity, Giardia trophozoites have an
efficient membrane trafficking system capable of directing numer-
ous cargo proteins to their correct destination along distinct trans-
port pathways (Marti et al., 2003a). This capability for constitutive
and stage-regulated secretion contrasts with the apparent simplic-
ity of the machinery involved and its molecular underpinnings:
although many conserved elements have been identified, global
analysis of the Giardia genome sequence revealed a significant
and consistent reduction of key components on every level
(Morrison et al., 2007). Most striking is the absence of certain
compartments or even entire organelles such as a Golgi apparatus
with biochemically defined cisternae and an endosomal system
that intersects with it, as well as key protein components such as
0020-7519/$36.00 Ó2011 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.ijpara.2010.12.014
⇑
Corresponding author. Tel.: +41 44 635 8526; fax: +41 44 635 89 07.
E-mail address: adrian.hehl@access.uzh.ch (A.B. Hehl).
International Journal for Parasitology 41 (2011) 471–480
Contents lists available at ScienceDirect
International Journal for Parasitology
journal homepage: www.elsevier.com/locate/ijpara
vesicle tethering factors. Combined with the induction of a prom-
inent secretory pathway in encysting cells, this makes Giardia a un-
ique biological model to investigate minimal solutions for
membrane transport.
2. Molecular machineries for trafficking
The description of the giardial membrane-bounded organellar
system relies both on morphology (Lindmark, 1988; Reiner et al.,
1989; McCaffery and Gillin, 1994; Lanfredi-Rangel et al., 1998;
Benchimol, 2004) and detection of common marker proteins
(Reiner et al., 1989; Marti et al., 2003b; Tovar et al., 2003; Touz
et al., 2004; Hernandez et al., 2007; Elias et al., 2008). The use of
heterologous antibodies to detect markers is problematic, how-
ever, due to the significant divergence of many giardial proteins.
This usually requires generation of specific antibodies against the
giardial homologs (Marti et al., 2003b; Elias et al., 2008). Although
the fine structures of the endomembrane system are yet to be de-
fined, the combined data reveals highly simplified organizational
principles on the one hand and highly specialized features on the
other. At morphological and molecular levels only three clearly
identifiable organellar systems have been delineated in trophozo-
ites: an extensive endoplasmic reticulum (ER) which is continuous
with the nuclear envelopes as in other eukaryotes, the PVs and
mitochondrial remnant organelles (mitosomes). An additional set
of prominent organelles, the encystation-specific vesicles (ESVs),
is generated only in encysting trophozoites and disappears again
upon secretion of CWM. Fig. 1 shows a surface-rendered composite
model of four identifiable organellar systems generated from a
three-dimensional reconstruction of a confocal image stack.
Several trafficking pathways to and from these organelles have
been identified and some are characterized in more detail. Current
evidence suggests that the ER (together with ESVs in differentiat-
ing cells) comprises the entire secretory system in Giardia.
Similarly, PVs are the only identifiable organelles involved in endo-
cytic transport. This is in line with the low number of conserved
members of key protein families involved in membrane transport
(see also below).
There is a growing body of evidence indicating a secondary loss
of major compartments and functions associated with membrane
transport in Giardia. However, the reason for this dramatic reduc-
tion is unknown. Nevertheless, Giardia still possesses core machin-
ery for membrane transport with the three well-known ubiquitous
eukaryotic coat complexes (COPII, coatomer (COPI), clathrin) and
two adaptor protein (AP) complexes. Interestingly, COPI and APs
are normally associated with the Golgi apparatus. All but one sub-
unit of the heteroheptameric COPI complex can be identified in the
Giardia genome. In addition, six small GTPases of the Arf and
Arf-like family of small GTPases which are involved in COPI recruit-
ment to membranes (Murtagh et al., 1992; Marti et al., 2003b)
were detected. At least one of the two giardial Arf1 homologs is
sufficiently conserved to rescue a lethal yeast Arf1/2 double mu-
tant (Lee et al., 1992). Giardia has only two complete hetero-tetra-
meric AP complexes corresponding to an AP1 and an AP2/3
equivalent; G. lamblia adaptor protein complex (AP1) is involved
in secretory transport (Touz et al., 2004), whereas the second com-
plex localizes to PVs and functions in endocytosis (Rivero et al.,
2010). The last common eukaryotic ancestor (LCEA) likely
contained three or four AP complexes which evolved early from a
proto F–COP–AP complex by coordinated gene duplication of their
subunits (Schledzewski et al., 1999; Boehm and Bonifacino, 2001;
Marti et al., 2003b) The presence of only two complexes in Giardia
again supports the secondary loss hypothesis. A giardial clathrin
Fig. 1. Surface-rendered composite model showing the compartment distribution and the morphology of the four Giardia organelles. The images were generated from a
three-dimensional reconstruction (volume image) of a confocal image stack using the Imaris software suite (Bitplane, Switzerland). The trophozoite is viewed from the
ventral side. Mitosomes or encystation-specific vesicles (ESVs) are shown together with the nuclei and the endoplasmic reticulum (ER) in A and D, respectively. (B) The
localization of clathrin. Note that clathrin is distributed around peripheral vesicle (PV) organelles and does not label the PVs directly. (C) A combined image with all four
organelle systems. The orientation of the model is indicated at the center of the image; the arrows point to the anterior (up), lateral (left) and dorsal (right) side of the cell.
Mitosome lumen marker: IscU (Giardia Genome Database GL50803_15196); ER membrane marker: protein disulfide isomerase 2 (PDI2) (GL50803_9413); PV region: clathrin
heavy chain (GL50803_9413); ESV lumen marker: cyst wall protein 1 (CWP1) (GL50803_5638); nuclear DNA: DAPI.
472 C. Faso, A.B. Hehl / International Journal for Parasitology 41 (2011) 471–480
heavy chain localizes to the PV region (Fig. 1) and to membranes of
maturing ESVs (Marti et al., 2003b; Gaechter et al., 2008) but a
light chain homolog was not identified. Whether this is the reason
for the lack of clathrin-coated transport intermediates and mem-
brane buds requires further investigation of clathrin coat
recruitment.
Rab (Ras-related in brain) GTPases, SNAREs (soluble N-ethyl-
malemide-sensitive factor attachment protein receptors), and their
effectors are universally conserved molecular machineries which
regulate and promote membrane fusion events and define com-
partment identities by conferring specificity. Due to the specific
interaction with membranes of organelles, their diversity is a good
indicator of the level of compartment complexity in a cell. Organi-
zation of the giardial trafficking pathways and compartment struc-
ture appears to require only seven Rab proteins (Marti et al.,
2003b) in contrast to the large Rab complements of other basal
protozoa such as Trypanosoma brucei (16 Rabs) (Ackers et al.,
2005), or the vastly expanded Rab families in Trichomonas vaginalis
(65 members) (Lal et al., 2005) and Entamoeba histolytica (>90
Rabs) (Saito-Nakano et al., 2005). Rabs consist mostly of a GTPase
domain and are functionally defined by a 30 amino acid domain
at their C-terminus, which includes prenylation sites. Thus, se-
quence constraints are high, which makes it likely that all giardial
Rabs have been identified. Three predicted exocytic (Rab1, 2a/
b)(Langford et al., 2002; Marti et al., 2003b) and one predicted
(recycling) Rab11 homolog (Castillo-Romero et al., 2010) are well
conserved, and cluster robustly with orthologues from other
eukaryotes in phylogenetic analyses. The remaining Rab family
members (F, D, and 32) cannot be assigned to specific Rab sub-
groups (Marti et al., 2003b; Morrison et al., 2007).
Consistent with experimental data indicating a simple compart-
ment structure with few trafficking pathways (Hehl and Marti,
2004), a small number of factors which mediate vesicle docking
and membrane fusion were identified. Members of the SNARE fam-
ily of proteins are highly divergent and therefore more difficult to
classify with confidence. Using BLAST search algorithms only seven
SNARE proteins (Dacks and Doolittle, 2002, 2004; Marti et al.,
2003b; Morrison et al., 2007) were initially identified in the com-
pleted genome. Three putative vesicle-(v-)SNAREs, and four syn-
taxin (target-(t-)SNARE) homologs were identified. Application of
more sensitive methods using Hidden Markov Model (HMM) pro-
files identified 15 (Kloepper et al., 2007) and 17 (Elias et al., 2008)
candidate SNARE family members, respectively. These studies pro-
vide excellent starting points for a detailed analysis of SNARE
function.
Long-range capture of transport vesicles before SNARE-driven
membrane fusion is mediated by large coiled-coil tethering factors
of the golgin family or generally conserved tethering complexes
such as GARP (Golgi-associated retrograde protein), COG (con-
served oligomeric Golgi) and exocyst. Because no Golgi stacks were
identified in Giardia it is not surprising that golgin family proteins
and other Golgi matrix proteins, which act as organizers of this
organelle, cannot be found in the genome database (Marti et al.,
2003b; Morrison et al., 2007). Exceptions are the TRAPPI (transport
protein particle) and the HOPS (homotypic fusion and vacuolar
protein sorting) complexes (Koumandou et al., 2007). Overall, de-
spite the conservation of several key factors and complexes, the
genomic analysis of the machinery mediating intracellular trans-
port shows consistent minimization.
3. Organelles and pathways for secretory transport
Secretory transport plays an essential role in Giardia: every life
cycle stage with the exception of the cyst outside of the host is
known to secrete specific soluble and membrane-bound proteins.
Accumulation of CWM in ESVs, and export beyond the plasma
membrane is the most conspicuous manifestation of secretory
transport in this parasite. Yet, maintenance of the protective VSP
coat alone likely requires a significant investment in secretory re-
sources. Export of both CWM and VSPs occurs simultaneously in
encysting cells, most likely until secretion of CWM. Thereafter,
VSPs are removed from the plasma membrane (McCaffery et al.,
1994; Marti et al., 2003a). These processes require distinct secre-
tory pathways, which do not intersect beyond the ER and are sub-
ject to stage regulation (Marti et al., 2003a). Thus, differentiating
parasites are forced to considerably increase their synthetic capac-
ity. One consequence is the de novo establishment of ESVs, an
additional set of secretory organelles dedicated to maturation
and export of the CWM.
3.1. Endoplasmic reticulum
The giardial ER can be visualized by immunofluorescence
microscopy using antibodies against resident proteins such as heat
shock protein 70/binding immunoglobulin protein (Hsp70/BiP) or
one of the five protein disulfide isomerases (PDIs) (Knodler et al.,
1999; Stefanic et al., 2006; Abodeely et al., 2009). The organelle ex-
tends from the nuclear envelopes through the entire cell body and
has an intricate, bilaterally symmetrical, structure. The very exis-
tence of this archetypical eukaryotic organelle in Giardia had been
called into question (Feely et al., 1990; Meyer, 1994), although ER
membranes had been identified by electron microscopy (Reiner
et al., 1990; McCaffery and Gillin, 1994). Definitive identification
of the giardial ER was achieved using antibodies raised against
the conserved giardial Hsp70/BiP homolog (Gupta et al., 1994;
Lujan et al., 1996b; Soltys et al., 1996). Confocal microscopy with
ER-Tracker™ and electron microscope (EM) tomography in living
trophozoites confirmed the general tubular nature and sub-cellular
distribution of the ER organelle (Abodeely et al., 2009). ER mem-
branes are found throughout the cytoplasm but do not permeate
the space occupied by PVs (Fig. 1). However, EM studies identified
apparent contact points and continuities between the two orga-
nelle systems. This is the basis for a recently postulated endocytic
transport hypothesis (Abodeely et al., 2009).
3.2. Glycosylation and ER quality control
While it appears that Giardia cells possess a conventional ER
with respect to secretory trafficking, some elements of the post-
translational modification machinery are missing entirely. Most
prominently, no calnexin–calreticulin machinery for quality
control of N-glycosylated secreted proteins has been identified
(Samuelson et al., 2005; Banerjee et al., 2007, 2008). This fits with
the absence of conventional GlcNAc
2
Man
9
Glc
3
core glycans linked
to asparagine (Asn) residues of proteins secreted from the ER
(Samuelson et al., 2005). Although complex branched glycans were
postulated (Morelle et al., 2005), rigorous genomic and biochemi-
cal analyses refuted this and revealed that Giardia misses several
nucleotide sugar transporters (Banerjee et al., 2008). In addition,
only one (i.e. Alg7) of the common 12 glycosyltransferases required
for synthesis of the dolichol-PP-GlcNAc
2
Man
9
Glc
3
precursor in the
ER (Samuelson et al., 2005) has been described. Thus, Asn-linked
glycosylation in the giardial ER is limited to the addition of
GlcNAc
1–2
to proteins. Nevertheless, a recent analysis revealed a
sizable N-glycome in Giardia with distinct aspects of stage regula-
tion (Ratner et al., 2008). Co-translational import and folding of se-
creted proteins is supported by a conserved machinery for
translocation (Svard et al., 1999), chaperones, and the five mem-
bers of the PDI family of proteins (Knodler et al., 1999; Morrison
et al., 2007). The latter are unusual in that they have a single thio-
redoxin domain instead of the normal two or three in PDIs of other
C. Faso, A.B. Hehl / International Journal for Parasitology 41 (2011) 471–480 473
eukaryotes. This domain structure appears to be a primary basic
feature and not the product of secondary reduction (Knodler
et al., 1999). Giardial PDIs most likely play a major role in assisting
the folding of the cysteine-rich VSPs, the high cysteine membrane
proteins (HCMPs) (Davids et al., 2006b) and cyst wall proteins
(CWPs).
3.3. Golgi-like organelles and Golgi functions
The earliest electron microscopic examination of cysts and tro-
phozoites revealed a conspicuous absence of organelles, specifi-
cally ER, Golgi and mitochondria (Sheffield and Bjorvat, 1977).
Later claims that a Golgi organelle existed (Gillin et al., 1991; Lujan
et al., 1995a; Lanfredi-Rangel et al., 1999; Dacks et al., 2003) re-
main unconfirmed. The classical Golgi is defined by a series of bio-
chemically distinguishable, dynamic, steady-state compartments
in which most or all secreted proteins are delayed for post-transla-
tional maturation before being sorted and transported to their final
destinations. Whether Golgi cisternae are arranged as easily
detectable ordered stacks with defined cis (receiving) and trans
(exporting) polarities is irrelevant since there are numerous exam-
ples of perfectly functional, delocalized Golgi systems. No known
Giardia organelle which meets these criteria has been identified
to date. Importantly, no classical markers for the Golgi such as
GM130, galactosyl transferases or the trans-Golgi network marker
Rab6 are present in Giardia. This constitutes a strong argument for
the loss of a stacked Golgi which was present in the LCEA (Dacks
and Field, 2007). Nevertheless, post-ER trafficking compartments
in Giardia exist in the form of ESVs of encysting trophozoites which
have many hallmarks of Golgi cisternae but also present clear dif-
ferences (see below). Components of the Golgi-associated COPI
were detected on ESV membranes (Stefanic et al., 2006). However,
antibodies against a giardial COPI subunit also label punctate
structures in trophozoites (Marti et al., 2003b). Indirect evidence
for the existence of post-ER trafficking compartments also derives
from the identification of a putative KDEL-receptor (Lys-Asp-Glu-
Leu) Erd2 homolog (GiardiaDB GL50803_4502) in the Giardia
genome. This protein is likely involved in retrieval of ER resident
proteins such as the chaperone Hsp70/BiP from distal compart-
ments (Stefanic et al., 2006).
4. Secretory transport during growth and differentiation
4.1. Secretion in trophozoites
The transmembrane-anchored VSPs of trophozoites are the ma-
jor surface proteins (Adam et al., 1988). VSP exodomains are re-
leased after cleavage in the conserved C-terminal domain
(Papanastasiou et al., 1996) and become soluble antigens. This re-
quires a constant turnover of VSPs at the surface, independent of
antigenic variation. Thus, synthesis and export of VSPs as well as
additional cysteine-rich, non-variable proteins (Davids et al.,
2006b) targeted to the plasma membrane likely constitute a major
part of the secretory activity of trophozoites. In addition to the
approximately 200 VSPs, more than 500 proteins with a signal se-
quence are currently predicted in the Giardia Genome Database
(www.giardiadb.org). An unknown number of these factors are ex-
ported to the surface or released into the environment by mem-
brane transport. Because Giardia lacks Golgi cisternae and delay
of secreted proteins in post-ER compartments during export has
not been observed (Marti et al., 2003a), secreted proteins are traf-
ficked directly to their final destination after leaving the ER. Cur-
rent data support direct transport of VSPs and likely of other
constitutively secreted soluble and membrane proteins from the
ER to the plasma membrane (PM) (Marti et al., 2003a; Touz
et al., 2003; Hehl and Marti, 2004). Interestingly, export of VSPs
is sensitive to brefeldin A (Lujan et al., 1995a), a fungal metabolite
which inhibits Arf1-dependent recruitment of COPI to Golgi mem-
branes and results in fragmentation of the Golgi organelle in higher
eukaryotes. This observation suggested that VSP transport is
dependent on COPI coat protein complexes, although no corre-
sponding post-ER compartment through which these proteins
transit was identified.
Few targeting signals have been characterized in secreted pro-
teins of trophozoites: short C-terminal sequences directing VSPs
to the PM and an encystation-specific protease (ESCP), (Touz
et al., 2002b) to PVs, respectively. The C-terminal domain of all
VSPs includes an invariable cytoplasmic tail CRGKA (Cys-Arg-Gly-
Lys-Ala), a hydrophobic transmembrane sequence, and a short,
well-conserved region of exodomain. The short cytoplasmic tail
is post-translationally modified by palmitoylation (Papanastasiou
et al., 1997; Touz et al., 2005) and by citrullination of the arginine
residue (Touz et al., 2008). The VSP C-terminal domain is necessary
and sufficient for correct secretion of a heterologous Toxoplasma
gondii SAG1 surface antigen exodomain (Kasper et al., 1984; Tom-
avo et al., 1992) used as a reporter in stably transfected trophozo-
ites (Marti et al., 2002). Removal of the CRGKA cytoplasmic tail
alone or of the entire C-terminal domain resulted in accumulation
of this reporter in the ER (Marti et al., 2003a), suggesting that this
sequence has an essential function in VSP targeting. On the other
hand, a different study showed that an intact VSP transmembrane
and exodomain is sufficient for trafficking to the PM in transgenic
cells (Touz et al., 2003). However, the possibility that the reporter
is co-exported by interaction with endogenous secreted VSPs in
these transgenic cells has not been tested.
ESCP is synthesized as a membrane-anchored pro-protein and
targeted to lysosomes in trophozoites (Touz et al., 2002b, 2003,
2004) by a conserved YXX
U
-type targeting signal YRPI (Tyr-Arg-
Pro-Ile) in its short cytoplasmic tail. Targeting of chimeric reporters
with a ESCP-derived transmembrane domain TM and cytoplasmic
tail and a H7 VSP exodomain to PVs was dependent on this motif
(Touz et al., 2003) and the AP complex 1(Touz et al., 2004). A Gol-
gi-like sorting function has been invoked for ESCP secretion; signif-
icant amounts of epitope-tagged ESCP on the cell surface (Touz
et al., 2003) could also be explained with YXX
U
-mediated retrieval
of ESCP from the PM.
The establishment of axenic culture systems (Keister, 1983) was
a prerequisite for the systematic identification of soluble secretory
products. In vitro (Katelaris et al., 1994; Roxstrom-Lindquist et al.,
2005; Muller et al., 2007) and rodent models (Davids et al., 2006a;
Li et al., 2007) for study of the effects of secreted substances on the
host are also well established. Research on secretion in trophozo-
ites focused on identifying virulence factors, i.e. products which
cause pathology in the host intestine or which interfere with
immunological reactions to giardial infections. No evidence for
the production of secreted toxins was found (Smith et al., 1982),
but a number of secreted proteins have been identified which
could modulate the physiology of host cells on different levels.
Unidentified secreted proteins were implicated in degradation of
barrier function and induction of apoptosis in epithelial cells after
co-cultivation with Giardia (Jimenez et al., 2000; Teoh et al., 2000;
Chin et al., 2002; Rodriguez-Fuentes et al., 2006; Panaro et al.,
2007; Troeger et al., 2007; de Carvalho et al., 2008). Other secretory
products affected the uptake of glucose and phenylalanine in the
intestine of mice (Samra et al., 1988).
Secreted (glyco)proteins appear to modulate certain aspects of
the intestinal immune response and may play a role in trophozoite
survival (Jimenez et al., 2007) although these claims require further
substantiation. In particular, an effect by contamination of serum
glycoproteins which tend to adhere to the trophozoite surface will
have to be excluded. In addition, substantial amounts of soluble VSP
474 C. Faso, A.B. Hehl / International Journal for Parasitology 41 (2011) 471–480
exodomains are released after cleavage in the conserved domain at
the base of the transmembrane anchor (Papanastasiou et al., 1996).
The effect of secreted VSPs, however, is unclear.
In addition to protein secretion via ER and membrane transport,
there are robust data suggesting an important role for non-conven-
tional protein export. Recently it was shown that direct contact be-
tween cultured intestinal epithelial cells and Giardia elicits distinct
changes in chemokine expression (Roxstrom-Lindquist et al.,
2005). Metabolic enzymes secreted by trophozoites (Ringqvist
et al., 2008) are able to alter the microenvironment in co-culture
assays. An example is the proposed depletion of arginine at the
host–parasite interface, resulting in suppression of nitric oxide
(NO) production (Eckmann et al., 2000). The pathway through
which the metabolic enzymes arginine deiminase, ornithine car-
bamoyl transferase and enolase are secreted upon contact with
epithelial cells in vitro is unknown (Ringqvist et al., 2008). These
exported effectors and immunogens (Palm et al., 2003) are cyto-
plasmic proteins, contain no N-terminal signal peptide and are
therefore not detected in vesicular transport pathways. Whether
export occurs through direct unconventional routes or if microve-
sicles such as exosomes (Silverman and Reiner, 2011) are involved
as shown for several prokaryotes and eukaryotes, is an interesting
question requiring further investigation.
Taken together there is solid support for Giardia interference
with epithelial function, presumably via soluble or membrane-
bound secreted proteins. However, these factors and their precise
interaction(s) with host cells await characterization.
4.2. Secretion in encysting cells
The most striking secretion process in the giardial life cycle oc-
curs during stage-differentiation of trophozoites. Secretion of the
CWM is the only known regulated export pathway. In fixed cul-
tured cells neogenesis of ESVs followed by deposition of this extra-
cellular matrix on the surface of differentiated cells is observed by
IFA (Gillin et al., 1987). Several protocols for in vitro encystation
are currently used in laboratories. The two-step method (bile
deprivation, followed by supplementation with porcine bile and
an increase of the pH to 7.85) (Boucher and Gillin, 1990) is the
most popular, but alternative methods such as cholesterol depriva-
tion (Lujan et al., 1996a) or incubation with high concentrations of
bile extract in the medium (Kane et al., 1991) are also very
effective.
Encysting trophozoites can be followed from induction to the
formation of the cyst wall. Although the time from induction to
cyst formation varies considerably between laboratories, this pro-
cess takes at least 14–20 h. Co-translational insertion of the struc-
tural proteins of the cyst wall, CWP1–3 (Lujan et al., 1995b;
Mowatt et al., 1995), into the ER takes place during the first 7h
post-induction (p.i.). The three CWP family members are soluble
proteins with a distinct structure, comprising a central domain
with several leucine-rich repeats (Lujan et al., 1995b). The CWM
biopolymer has a surprisingly low complexity considering how
effectively it acts as a biological barrier on the surface of the cyst.
The three CWPs are paralogous and constitute approximately 40%
of the cyst wall; the rest of this extracellular matrix is made up of a
simple b1-3 linked N-acetylgalactosamine (GalNAc) homopolymer
(Jarroll et al., 1989; Gerwig et al., 2002). A non-VSP type 1 integral
membrane protein termed HCNCp is a member of a large group of
cysteine-rich factors which are potentially secreted stage-specifi-
cally (Davids et al., 2006b). Indeed, while an epitope-tagged variant
of the HCNCp investigated in this study localized to the plasma-
lemma in trophozoites, it was detected in ESVs during encystation
and partially secreted to the cyst wall or the PM – cyst wall inter-
face (Davids et al., 2006b). Recently, another family of soluble cys-
teine-rich proteins which appear to be trafficked via ESVs to the
cyst wall but localize additionally to the cell body have been iden-
tified (Chiu et al., 2010). On the other hand, CWPs are trafficked
exclusively through ESVs and are secreted quantitatively. How-
ever, it is not known when and where in this exocytic pathway
the poly-GalNAc sugar component is synthesized and how it is fi-
nally incorporated into the cyst wall structure. A recent study
showed that the leucine-rich repeat domain of CWP1 has a lectin
activity and binds to the curled fibrils of the GalNAc homopolymer
in the cyst wall (Chatterjee et al., 2010). Taken together with the
sequential secretion of the CWPs (see below) this suggests that
the glycan and at least CWP1 are exported coordinately either in
the same or separate carriers and polymerize after distribution as
an even layer on the surface. Time-lapse microscopy of living cells
showed that the deposited CWM loses its plasticity and transforms
into a rigid shell in 10–30 s (Trepp, F., Spycher, C., Hehl, A.B.,
unpublished data).
The synthesis and trafficking of the CWM components are
stage-specifically regulated and highly coordinated. The assembly
of the cyst wall galactosamine polymer from glucose is mediated
by pathways whose components are upregulated transcriptionally
and allosterically after induction (Macechko et al., 1992; Lujan
et al., 1995a; Das and Gillin, 1996; Van Keulen et al., 1998; Bulik
et al., 2000). CWP1–3 mRNA levels peak at 7 h p.i., and the newly
produced CWPs accumulate at approximately 2 h p.i. in emerging
ESVs (Konrad et al., 2010). CWP export from the ER to ESVs is com-
pleted 8–10 h after induction (Hehl et al., 2000). The fact that ESVs
only contain CWPs and no constitutively secreted proteins strongly
suggests that the former are sorted away from the latter during ER
export (Reiner et al., 1990; Marti et al., 2003a). The first half of the
encystation process is therefore dedicated to synthesis and accu-
mulation of the CWM in the newly established ESVs. This sorting
and export pathway in the absence of a constitutive Golgi appara-
tus has hallmarks of cis Golgi compartment neogenesis. The mem-
brane carriers, if any, for trafficking of the CWM from the ER to
ESVs are currently unknown, but the process appears to be depen-
dent on COPII coat formation and the small GTPase Rab1 (Stefanic
et al., 2009). An alternative scenario invokes lateral segregation of
the CWM into ER sub-domains which eventually differentiate into
ESVs (Lujan and Touz, 2003; Hehl and Marti, 2004). Segregation of
the CWPs to ESVs is determined by dominant signals on CWPs and
occurs at the level of ER export (Marti et al., 2003a). Rather than
defined targeting sequences, entire domains of CWPs, specifically
the leucine-rich repeats (LRRs), are necessary for sorting into ESVs
(Hehl et al., 2000; Marti et al., 2003a; Sun et al., 2003). Thus, sim-
ilar to Golgi cisternae, ESVs are post-ER organelles where cargo is
delayed, presumably for maturation, before regulated secretion.
However, unlike conventional Golgi cisternae, ESVs contain only
one type of cargo, the CWM. However, transient association of COPI
components with ESVs (Marti et al., 2003b), their sensitivity to bre-
feldin A (Lujan et al., 1995a; Marti et al., 2003a), and dependence of
ESV genesis and maturation on giardial Sar1 and Arf1 GTPases,
respectively (Stefanic et al., 2009), support the idea that these
organelles are stage-regulated Golgi-like compartments. The exact
nature of ESVs and their function in regulated secretion in the con-
text of reductive evolution are currently under investigation in sev-
eral laboratories.
After completion of ESV formation CWP1, which is used as a
standard marker for the CWM, is further delayed in ESVs for several
hours, before being secreted to cover the entire cell surface where it
eventually polymerizes. Recent studies revealed that the formation
of an environmentally-resistant cyst wall is more complicated and
requires several tightly synchronized steps (Gottig et al., 2006; Stef-
anic et al., 2009; Konrad et al., 2010). The three CWP family mem-
bers share considerable sequence identity and have a similar
domain structure. CWP2 has an additional 121 residue C-terminal
extension rich in basic amino acids (Lujan et al., 1995b). Proteolytic
C. Faso, A.B. Hehl / International Journal for Parasitology 41 (2011) 471–480 475
processing of this domain is currently the only proteolytic modifi-
cation described for CWPs during maturation of the CWM. In addi-
tion, formation of disulfide (Hehl et al., 2000) and isopeptide
(Davids et al., 2004) bonds between CWPs appears to play a major
role in the export process. Although the evidence clearly implicates
specific cleavage of pro-CWP2 by a cysteine protease, controversy
as to the exact identity of the enzyme remains (Touz et al., 2002a;
DuBois et al., 2008). Previous investigations indicated that the en-
tire C-terminal extension of 13 kDa (Gottig et al., 2006) might
be removed from CWP2. However, the small C-terminal portion of
the native or the transgenic CWP2 had not yet been visualized di-
rectly (Sun et al., 2003). Although processing of CWP2 occurred be-
fore secretion of the CWM, its correlation with expression kinetics
or maturation and morphology of ESVs had not been explored until
recently. Analysis of a CWP2 variant with epitope tags at both ter-
mini showed that cleavage occurred during the maturation stage of
ESVs, before secretion of the CWM (Konrad et al., 2010). Western
blot analysis suggested removal of a short fragment of 5 kDa be-
tween 8 h and 10 h p.i. The C-terminal domain of CWP2 had been
implicated in sorting of all three CWPs from the ER to ESVs (Gottig
et al., 2006). The authors had invoked an export mechanism based
on sequestration to and protrusion from specialized sub-domains of
the ER, rather than conventional ER export. The relation between
ESVs and the ER remains unclear. On the one hand there is a distinct
morphological difference between the two organellar systems as
well as exclusion of cargo and resident factors such as PDIs and
Hsp70/BiP from ESVs. The latter, however, appears to be cycling
through ESVs and may be retrieved by a KDEL receptor protein
(Stefanic et al., 2006). On the other hand, the current data give no
answers to the questions as to how ESVs arise and whether the nas-
cent organelles at least constitute highly specialized ER sub-do-
mains (Touz et al., 2002a; Marti et al., 2003a; Gottig et al., 2006).
In particular, the close spatial proximity of the two organelles and
the vigorous exchange of the CWM between established ESVs raises
the question whether they ever become truly independent before
the secretory process begins (Stefanic et al., 2009).
Dual-tagged CWP2 as well as CWP::GFP chimeras to analyze the
processes during the maturation phase of ESVs revealed a hitherto
unnoticed sorting and partitioning of the CWM (Konrad et al.,
2010). In particular, co-localization studies of CWP1 with CWP3
or with the C-terminus of CWP2 showed that the CWM is parti-
tioned into two biophysically distinct fractions inside maturing
ESVs. CWP3 forms a condensed core structure together with the
small CWP2 C-terminus after processing of pro-CWP2. How the
condensation process is triggered is unknown but an unrelated,
earlier study showed that expression of giardial CWP1 and CWP2
in human embryonic kidney-293 cells led to formation of granules
and secretion to the culture medium (Abdul-Wahid and Faubert,
2004). This suggests that accumulation and condensation is an
inherent property of CWPs. In particular, the basic C-terminal
extension of CWP2 had been implicated in granule formation dur-
ing encystation in this study. Conversely, in encysting Giardia
CWP1 and the mature N-terminal, CWP2 remain in a fluid state un-
til secretion and distribution on the surface of the forming cyst
(Konrad et al., 2010). Thus, the condensed and the fluid fraction
of the CWM contains a mature CWP2 product. Live cell analysis
using GFP-tagged CWP1 variants showed that these cargo compo-
nents are highly motile within an ESV organelle network. These
studies demonstrate that the immobile ESVs are laterally con-
nected via dynamic membrane tubular channels (Stefanic et al.,
2009; Konrad et al., 2010). After partitioning of the fluid and con-
densed fraction of the CWM, the former is sorted away into com-
partments which localize close to the cell periphery. The upshot
is that this fluid material containing CWP1 and the large N-termi-
nal fragment of the processed CWP2 is rapidly and quantitatively
secreted within a few minutes. This first layer of the cyst wall is
laid down at the same time as morphological transformation of
the differentiating cell takes place. The material polymerizes rap-
idly and results in the formation of a structurally resistant cell wall.
The condensed rest of the CWM (CWP3 and the small C-terminal
fragment of CWP2) remains completely in internal compartments
and is secreted slowly over the course of several hours. Although
processing of CWP2 is not required for condensed core formation
and sequential secretion of CWM, it is necessary for correct parti-
tioning of CWP2. Unprocessed CWP2 becomes sequestered in the
condensed cores and is exported only during the second secretion
process resulting in the formation of morphologically normal cysts
which are not water-resistant (Konrad et al., 2010).
Taken together, regulated secretion and cyst wall formation ap-
pears to be significantly more complex than previously thought
and requires several hours from the time the first layer is estab-
lished until the cyst becomes water resistant. Condensed core for-
mation in maturing ESVs and sorting of the CWM for sequential
secretion is strikingly similar to processes at the trans-Golgi net-
work. This lends additional support to the hypothesis that ESVs
are functional Golgi cisternae analogs.
4.3. Secretion in excysting cells
When passing through the low pH environment of the stomach
Giardia cysts are triggered to initiate excystation. Secreted prote-
ases released from the endosome–lysosome PV system play a brief
but essential role in the liberation of the excyzoite (Bernander
et al., 2001) from the protective confines of the cyst wall. Host
and parasite proteases are required for the degradation of the
CWPs whilst a parasite-specific glycohydrolase attacks the GalNAc
fibrils of the cyst wall (Chatterjee et al., 2010). A cathepsin B type
protease (Ward et al., 1997), designated CP2, was identified by
affinity purification with a biotinylated variant of the specific
inhibitor E-64. The inhibitor was also used to determine the sub-
cellular localization of CP2 in PVs or the equivalent of these organ-
elles in cysts. Secretion of the enzyme into the space between the
plasma membrane and the cyst wall is triggered during
excystation.
CWPs are phosphorylated during export (Slavin et al., 2002).
This post-translational modification needs to be reversed for
excystation: inhibition of acid phosphatase activity during the first
phase of in vitro excystation almost completely abolished the pro-
cess (Slavin et al., 2002).
5. Organelles and pathways for endocytic transport
The PVs constitute a very conspicuous organelle system in
transmission EMs of trophozoites. The electron lucent PVs appear
approximately oval-shaped and typically 150 nm long. The
organelles are arrayed as a layer just below the plasma membrane
of the entire dorsal side as well as in the small region at the center
of the ventral disk. In the absence of a cytostome, the PVs are the
only known endocytic organelles (Tai et al., 1993) capable of accu-
mulating fluid phase and membrane-bound molecules. However,
PVs also seem to have lysosomal properties (Feely and Dyer,
1987; Lindmark, 1988; McCaffery and Gillin, 1994), which means
they acidify and mature to become digestive organelles. In addition
to hydrolases, PVs contain cathepsins (Ward et al., 1997; Thirion
et al., 2003). The presence of CWPs in the lumens of PVs (Reiner
et al., 1990) also suggests a role in regulated secretion of CWP.
The dynamics of fluid phase and membrane cargo uptake was
not investigated until recently. Two studies presented data sug-
gesting that PVs periodically open to the environment either via
a channel or by fusion with the PM and take up soluble material
(Gaechter et al., 2008; Abodeely et al., 2009) before closing again.
476 C. Faso, A.B. Hehl / International Journal for Parasitology 41 (2011) 471–480
The reversible, transient communication with the extracellular
space effectively flushes the PV, releasing its contents into the
environment and replacing this volume with extracellular med-
ium. Using fluid phase markers and confocal live-cell imaging it
was possible to determine the dynamics of this environmental
sampling and to quantify endocytic transport. PVs in living and
in chemically fixed cells can also be visualized using Lysotracker™
(Gaechter et al., 2008; Rivero et al., 2010). More importantly, fluo-
rescence recovery after photobleaching (FRAP) demonstrated that
there is no lateral exchange of fluid phase markers between indi-
vidual PVs (Gaechter et al., 2008). Most fluid phase markers re-
mained in PVs until expelled when the organelle again formed a
continuity with the external medium, but others (e.g. casein)
translocated rapidly to the ER or an associated membrane com-
partment termed the tubulo-vesicular network (TVN) (Abodeely
et al., 2009). These data suggest that uptake of soluble material
from the environment into PVs is not selective, in contrast to fur-
ther retrograde transport which allows only certain, as yet unde-
fined substances, to rapidly cross over into the proximal ER
(Abodeely et al., 2009). Most of the fluid phase material taken up
by PVs is likely to require further breakdown after acidification
of the organelle lumen before being transported along endocytic
pathways. A recent study shows that import of low-density lipo-
protein (LDL), which is not present in the gut lumen but abundant
in cell culture medium, is mediated by PVs and dependent on the
predicted AP 2/3 complex (designated as AP2 by the authors)
(Rivero et al., 2010).
PVs may therefore constitute a clever environmental sampling
system which allows the parasite to take up fluid phase material
relatively indiscriminately but safely. Subsequent digestion liber-
ates essential building blocks for transport towards the cell interior
whereas waste material as well as harmful substances are expelled
rapidly.
6. Mitochondrial remnants
Giardia lamblia has long been considered a primitive eukaryote
based on the absence of organelles resembling mitochondria. Based
on the Archezoa hypothesis, this organism was assigned to a loosely
classified group of supposedly amitochondriate lineages that had
not participated in the endosymbiotic event which led to the incor-
poration, maintenance and molecular ‘‘surrender’’ of an
a
-proteo-
bacteria (Cavalier-Smith, 1998). This theory concerning
eukaryotic speciation was strongly challenged by the discovery of
highly degenerate mitochondrion-related organelles in E. histolytica
named mitosomes (Tovar et al., 1999). Shortly thereafter,
G. lamblia homologs of genes IscS and IscU involved in the biosyn-
thesis of Fe–S proteins (an essential function of mitochondria) were
cloned, and their products characterized in terms of sub-cellular
localization in giardial cells (Tovar et al., 2003). Both proteins were
found to accumulate within double membrane-bound organelles
containing no DNA, as previously observed with E. histolytica.
In confocal microscopy images, Giardia mitosomes appear as
100 nm spherical cytoplasmic compartments. An intriguing
aspect of these organelles is their distribution into two distinct
populations, namely an apparently elongated single central mito-
some (CM) organelle and the multiple peripheral mitosomes
(PMs) (Regoes et al., 2005). PMs appear to be distributed randomly
throughout the cell while the CM is invariably found in the basal
body region between the nuclei. A more detailed ultrastructural
analysis by EM revealed that the CM was in fact a tight cluster of
spherical organelles which are indistinguishable from the PMs
(Hehl et al., 2007). This special configuration almost certainly has
a functional significance and was termed the central mitosome
complex (CMC). Upon cell division, only the CMC was shown to
be actively divided and segregated in a cell cycle-dependent man-
ner whilst the PMs appear to be distributed stochastically between
daughter cells (Regoes et al., 2005). The mechanisms which regu-
late mitosomal division and inheritance remain to be elucidated.
In an effort to further characterize giardial mitosomes, a careful
investigation of cellular distribution using specific antibodies
proved that other mitochondrial markers such as the giardial
homologs of chaperonin 60 (Cpn60), mitochondrial Hsp 70
(mtHsp70) and ferredoxin (Fd) were also present in these organ-
elles (Regoes et al., 2005). Taken together, these data indicate that
the absence of bona fide mitochondria in G. lamblia is the result of
secondary loss and not a manifestation of a primary primitive
state.
Interestingly, only giardial iron–sulfur cluster assembly enzyme
(IscU) and Fd were shown to require the presence of amino-termi-
nal targeting presequences, suggesting that mitosomes have main-
tained aspects of both presequence-dependent and independent
import routes (Tovar et al., 2003; Regoes et al., 2005). In the at-
tempt to investigate the molecular machinery mediating protein
import across the double mitosomal membrane, a component of
the inner mitochondrial membrane translocase and the processing
peptidase were identified in a comparative study involving Giardia
mitosomes and T. vaginalis hydrogenosomes (Dolezal et al., 2005).
Combined with the recent identification of a giardial Tom40 sub-
unit of the outer membrane protein import channel (Dagley
et al., 2009), this data supports the hypothesis that mitosomes,
hydrogenosomes and mitochondria represent different forms of
the same fundamental organelle and employ similar translocation
mechanisms for mitosomal protein import (Burri and Keeling,
2007). Further research will be necessary to characterize all com-
ponents of the translocation complexes to comprehend if and
how proteins with and without a canonical targeting presequence
are substrates for the same translocation machinery.
Thus, the identification of mitosomes as mitochondrial rem-
nants further reinforces the notion that extreme lifestyles, fre-
quently encountered in parasitic protozoa such as G. lamblia, may
lead to a substantial reduction in molecular machinery. This in turn
could result in the complete loss of entire organelle types such as
the Golgi apparatus and mitochondria.
7. Conclusions
The secretory transport capacity of Giardia is perfectly adapted
to a changing environment and able to deploy essential protective
surface coats as well as molecules which act on the cells of the host
epithelia. Nevertheless, the giardial trafficking machinery appears
to be the product of a general secondary reduction process that
led to minimization of all components identified to date (Marti
et al., 2003b; Morrison et al., 2007). Despite the low complexity
of the organelles and machinery involved, these diverse protective
surface antigens, as well as secreted proteins, are delivered with
great fidelity (Reiner et al., 1990; McCaffery et al., 1994; Hehl
et al., 2000; Marti et al., 2002, 2003a; Lujan and Touz, 2003; Touz
et al., 2003; Hehl and Marti, 2004; Gottig et al., 2006; Hernandez
et al., 2007; Konrad et al., 2010; Rivero et al., 2010). Of particular
interest are VSPs of trophozoites and the extracellular matrix poly-
mer of cysts, both of which confer environmental resistance in the
respective life cycle stages. Recent and ongoing work in several
laboratories is revealing the basic principles behind the transport
events responsible for the assembly of these key biological barriers.
However, significant work to elucidate the details of these mecha-
nisms, in particular in light of their astonishingly low complexity,
is still needed. For example, it is becoming clear that Giardia could
serve as a model for the investigation of synthesis, transport and
assembly of simple but highly effective biopolymers, a key feature
C. Faso, A.B. Hehl / International Journal for Parasitology 41 (2011) 471–480 477
of all perorally transmitted protozoan and metazoan parasites
(Gottig et al., 2006; Chatterjee et al., 2010; Konrad et al., 2010). An-
other example is the PV system: these organelles appear to be at
the crossroads of endocytic and exocytic transport. Although the
data are still scarce, PVs are emerging as the major sorting station
for the transport of soluble as well as membrane-bound factors in
and out of the cell (Touz et al., 2004; Abodeely et al., 2009; Rivero
et al., 2010). As such, they could provide a protected space for both
final modification and/or activation of secreted molecules on one
hand; on the other hand, PVs can act as an intracellular contain-
ment system. This would allow controlled separation of nutrients
from potentially harmful substances taken up in bulk by constant
sampling of the fluid extracellular environment. As is also the case
for ESVs, a better understanding of the biological role of trafficking
pathways to and from PVs requires elucidation of the ‘‘organelle
cycle’’, i.e. the processes that govern organelle genesis, mainte-
nance and maturation.
Acknowledgements
We apologize to the colleagues whose work we could not cite
for reasons of space. The work in the laboratory of ABH is sup-
ported by the Swiss National Science Foundation (grant
#31003A-125389). We are grateful to all current and past mem-
bers of the laboratory for their contributions, in particular to Dr.
Attila Regoes (University of Zürich, Switzerland) for providing the
composite volume image. We thank the reviewers of this article
for their helpful comments.
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