Fact 1: Infection with Giardia lamblia is one of the most common causes of waterborne
nonbacterial and nonviral diarrheal disease
G. lamblia (syn. intestinalis, duodenalis) is a zoonotic enteroparasite. It proliferates
in an extracellular and noninvasive fashion in the small intestine of vertebrate hosts,
causing the diarrheal disease known as giardiasis. Virtually all mammals can be infected
with G. lamblia, and epidemiological data point to giardiasis as a zoonosis [1]. Infections
in humans may be asymptomatic or associated with diarrhea, malabsorption, bloating,
abdominal pain, fatigue, and weight loss. Based on the latest figures provided by
WHO, G. lamblia is the third most common agent of diarrheal disease worldwide with
over 300 million reported cases per annum, preceded only by rotavirus and Cryptosporidium
parvum and hominis in the most vulnerable target group of children under five years
of age [2]. The prevalence of giardiasis in humans ranges from 2%–3% in industrialized
countries, up to 30% in low-income and developing countries [3]. Giardiasis was formerly
included in the WHO neglected diseases initiative and is directly associated with
poverty and poor quality of drinking water [4]. Acute infection develops over a period
of three weeks, peaking at eight days post infection. Generally, healthy hosts clear
the infection within 2–3 weeks, whereas the occasional chronically infected host shows
signs of villus and crypt atrophy, enterocyte apoptosis, and ultimately severe disruption
of epithelial barrier function [5]. Infection with G. lamblia has also been linked
to the development of irritable bowel syndrome and chronic fatigue [6].
Fact 2: G. lamblia presents a simplified subcellular organization but is not a primitive
eukaryote
To date, four types of endomembrane compartments have been identified in the Giardia
trophozoite, namely: the endoplasmic reticulum (ER), the nuclei, terminally-differentiated
mitochondrial remnants named mitosomes, and peripheral vacuoles (PVs) [7]. Encystation-specific
vesicles (ESVs) constitute a fifth compartment present only in encysting cells. Extreme
genomic divergence has led to frequent artefacts such as long-branch attraction in
earlier phylogenetic studies [8]. Combined with observations of elements of prokaryotic
metabolism and the absence of bona fide eukaryotic organelles such as the Golgi apparatus,
endosomes, and mitochondria, this resulted in a misclassification of G. lamblia as
a primitive eukaryote and a concomitant misinterpretation of its evolutionary history
[9]. However, molecular paleontology approaches aimed at identifying machinery present
in the last eukaryotic common ancestor (LECA) indicated that this organism likely
possessed all extant eukaryotic organelles and corresponding trafficking pathways.
This supports the notion that species with a simplified cellular organization such
as the ancestor of G. lamblia evolved via a reduction of complexity, likely linked
to adoption of a parasitic lifestyle, and that G. lamblia is therefore most likely
not a primary primitive eukaryote but secondarily reduced. Interestingly, recent efforts
aimed at rooting the eukaryotic tree place this root between the Excavata supergroup,
to which G. lamblia belongs, and all other eukaryotes [10].
Fact 3: G. lamblia feeds using specialized organelles called PVs
Due to streamlining of most anabolic pathways, G. lamblia cells are highly dependent
on nutrient uptake from the host’s gut by means of an array of organelles, i.e., PVs
(Fig 1A). This active host–pathogen interface is at the crossroads of both endo- and
exocytic trafficking in G. lamblia trophozoites (Fig 1B). The main function of PVs
is to periodically endocytose fluid-phase extracellular material and to expel harmful
or unusable substances into the environment again. This is in contrast to the unidirectional
endocytic uptake of fluid-phase material via cytostome-like structures in many protozoa
as well as in Spironucleus spp., the closest known relatives of Giardia [11]. PVs
effectively act as “safety-lock" compartments for efficient environmental sampling,
i.e., the uptake and intracellular sorting of gut content. The “kiss and flush” working
model (Fig 1C) developed in our group is based on experimental data suggesting that
PV membranes and the plasma membrane (PM) transiently fuse (the “kiss” phase) and
become continuous, thereby generating an opening to the extracellular space allowing
exchange of fluid-phase material between the PV lumen and the environment [12]. This
PV–PM connection is then resolved, and sorting of usable nutrients occurs within the
acidifying PV lumen. Any material that is not retained would then be released back
into the extracellular space in a new round of PV–PM connection (the “flush” phase).
The flushing of the PV lumen makes exchange of fluid-phase material bidirectional
and likely compensates for the lack of bona fide lysosomes as endpoints of endocytic
transport. Endocytosis through PVs is likely the main route of nutrient uptake into
the Giardia cell, although there are isolated reports on receptor-mediated uptake
of lipid particles. The giardial putative low-density lipoprotein (LDL) receptor (GILRP;
purple ribbon in Fig 1C) in G. lamblia was shown to interact with AP2 components [13],
although its exact trafficking mechanism remains uncharacterized (Fig 1C). Short actin
filaments were also shown to be involved in LDL uptake and were localized in close
proximity to PVs [14].
10.1371/journal.ppat.1007250.g001
Fig 1
A working model for fluid-phase and receptor-mediated nutrient uptake through the
PVs of G. lamblia.
(A) A TEM image of a PV making contact with a PM-derived invagination (white star).
(B) Connections between PVs and ER membranes (white star) are frequently detected
in TEM tomograms. (C) The “kiss and flush” working model for fluid-phase and receptor-mediated
endocytosis in PVs is based on previously published data [12,13,14,27] and is represented
as a continuum of nutrient entry, retention, release, and transfer. (1) Acidifying
PVs contain fluid-phase cargo, which is either free or retained by PV-resident receptors
(blue ribbons). Released cargo travels further to the lumen of connecting ER tubules.
Some cargoes (green pentagon) can be excluded from further passage to the ER. (2)
Uptake is mediated by fusion between the VSP-coated PM and the PV membrane through
PM-derived invaginations surrounded by clathrin arrays associated to AP2 complexes.
This event corresponds to the “kiss” phase in which formation of a channel allows
exchange of fluid-phase material between the PV lumen and the extracellular space.
Usable nutrients may move freely or be bound by receptors lining the organelle lumen,
whereas useless or harmful molecules may be released back in the extracellular space
during the “flush” phase. LDL receptor (GILRP) traffics in an AP2-dependent manner
from the PM (1) to the PV membrane. (3) PV–PM luminal continuity terminates and a
new round of receptor-bound nutrient release mediated by intralumenal acidification
ends with further passage to connected ER. (4) The PV is now ready for another round
of endocytosis and exchange with the extracellular environment and GILRP is recycled
back to the cell’s surface. ER, endoplasmic reticulum; GILRP, giardial putative low-density
lipoprotein receptor; LDL, low-density lipoprotein; PM, plasma membrane; PV, peripheral
vacuole; TEM, transmission electron microscopy; VSP, variant surface protein.
Fact 4: G. lamblia survives in the environment as infectious cysts
Completion of the life cycle by transmission of G. lamblia to a new host requires
no vectors and is based on the alternation of a vegetative stage, the trophozoite,
and an environmentally resistant infectious stage—the cyst. The cyst is the only stage
of G. lamblia able to survive outside of the host and is responsible for the initiation
of a new infectious cycle. Cyst development may already begin in the small intestine
of parasitized hosts when a variable fraction of proliferating trophozoites initiates
a cellular differentiation program called encystation [15]. Laboratory protocols for
inducing encystation include lipid depletion and an increase in culturing medium pH
over a period of ca. 20–24 hours [16]. The assumption is that these conditions mimic
decreasing lipid availability and ascending pH gradients naturally present along the
gastrointestinal tract. During encystation, flagellated pear-shaped binucleated trophozoites
undergo dramatic morphological and biochemical cellular remodeling, culminating in
the formation of nonflagellated oval quadrinucleated cysts, surrounded by a cyst wall
(CW). The CW is composed of a thick mesh of cyst wall proteins (CWPs) complexed to
a unique sugar polymer of β1,3-linked-N-acetylgalactosamine [17] and virtually shields
the cyst’s interior from any solvent. Deposition of the CW is a tightly-regulated
event that occurs exclusively in encysting trophozoites and requires neogenesis of
specialized secretory organelles called ESVs [18]. ESVs traffic, sort, and modify
mainly CWPs from their initial site of deposition at the ER en route to the parasite
cell’s surface. Elimination of a single CWP by complete gene disruption was shown
to abolish CW formation altogether [19]. The exact in vivo stimuli for this process
are not yet well known, although recent studies on the dynamics of encystation in
animal models point towards a link between high-density focal trophozoite populations
in the proximal small intestine and encystation [15]. These findings argue against
the natural pH and lipid gradients being the sole external triggers for differentiation.
In turn, this raises the interesting possibility that trophozoites can sufficiently
alter the local environment to generate conditions favorable for triggering differentiation.
Cysts are then shed through host feces and were reported to remain viable for several
months in water at temperatures below 10 °C and several weeks at room temperature
[20]. Based on experimental gerbil (Meriones ungulatus) infections, the minimal infectious
dose is less than 10 cysts [21]. Once viable cysts are ingested, passage through the
stomach and physical stimuli (temperature, pH) initiate a cellular program termed
excystation in which both host and parasite proteases collaborate to degrade the CW,
allowing the short-lived excyzoite to escape and rapidly divide twice to give rise
to four trophozoites [22]. In vitro (and most likely in vivo) this process is completed
within minutes [23].
Fact 5: Novel rational design-based vaccination strategies against G. lamblia are
yielding encouraging results
Currently, treatment of giardiasis in humans is based almost exclusively on administration
of antiprotozoals belonging to the family of 5-nitroimidazoles, whereas infected animals
are treated with benzimidazoles. A crude veterinary vaccine called GiardiaVax for
nonchronically infected dogs and cats was already licensed [24]. However, it does
not inhibit trophozoite proliferation and in many cases has to be combined with additional
drug treatment. In recent years, work done especially by the Lujan group at the Universidad
Católica de Córdoba in Argentina has uncovered the potential for variant surface proteins
(VSPs) as effective vaccination antigens in companion animals. The entire trophozoite
surface is covered by a dense coat of VSP anchored in the plasma membrane with a C-terminal
hydrophobic transmembrane anchor sequence. From a repertoire of over 200 homologous
genes encoded in the parasite genome, only one VSP is expressed on the surface of
every single trophozoite at any given moment. Antigenic switching and regulation of
VSP expression were shown to occur by RNAi-like mechanisms that could be perturbed
to deregulate VSP production, leading to trophozoites exposing the full VSP panel
on their surface [25]. Oral vaccination trials on gerbils showed that animals initially
infected with deregulated cells expressing all of the VSPs encoded in their genome
are largely protected from challenge infections by Giardia clones that express a unique
VSP on their surface or by cysts obtained from infected individuals [25]. The same
vaccine was tested on cats and dogs, showing high efficiency in preventing new infections
and reducing chronic giardiasis in domestic animals both in experimental and natural
infections [26]. These data are based on the rational design of vaccination strategies
underpinned by a deep understanding of G. lamblia’s molecular and cell biology and
hold great promise for eradication of giardiasis in both animals and humans.