Across intra-mammalian stages of the liver f luke Fasciola hepatica: a proteomic study

Across intra-mammalian stages of the liver f luke Fasciola hepatica: a proteomic study

Across intra-mammalian stages of the liver f luke Fasciola hepatica: a proteomic study

Giardia lamblia is a deeply branched zoonotic intestinal protozoan pathogen. It infects humans, causing the diarrheal disease giardiasis, throughout the world (1, 2). Prolonged infections
in humans often cause malabsorption of nutrients with weight
loss in the absence of treatment, despite an immune response that
would be expected to control the infection (3). Both innate and
adaptive immune responses are apparently involved in giardiasis
(4, 5).

Across intra-mammalian stages of the liver f luke Fasciola hepatica: a proteomic study
Across intra-mammalian stages of the liver f luke Fasciola hepatica: a proteomic study

In a likely defense to the host immunity, antigenic variation,
a mechanism known for many pathogens to evade the humoral
immune response of their vertebrate hosts (5), was shown to occur
on the membrane surface ofGiardia trophozoites both in vitro and
in vivo (6, 7). Individual Giardia trophozoites express only a single
species of variant-specific surface protein (VSP) on the surface of
the cell membrane at any given time (8) and subjects to replacement by another VSP maxanim every 6 to 13 generations of cell growth (9).

The first VSP was identified in the trophozoites of a Giardia
WB isolate from a partial mRNA sequence encoding a 170-kDa
protein (initially named CRP170 but later designated VSPA6),
which was recognized on the cell membrane surface by a 6E7

The N terminus of VSP cannot be blocked without affecting
its cell membrane localization. Although the N domain of VSP175(VSPA6-S1) does not participate in localizing the latter to the
membrane surface (Fig. 6D), our previous data in Fig. 2A and B
indicated that tagging the N terminus of VSP9B10A with 3 myc
resulted in a failure of its expression on the membrane surface.

Other previous observations that an N-terminal ~14- to 17-
amino-acid sequence was missing from TSA417 and VSPH7 when
they were isolated from cell membranes suggested that the
N-terminal sequence could function as a targeting signal for translocalization and become removed after completion of the process

In view of the highly varied N-terminal sequences among
the verified VSPs and the localization of motif 1-link-motif 2 of
VSP175 to the membrane surface without an N domain (Fig. 6F),
there is the possibility that a specific N-terminal targeting signal
may not be needed for VSP translocalization. To clarify this possibility further, VSP-175(VSPA6-S1), VSP-98.1(VSP1267), VSP312, and VSP-338 were each tagged with 3 myc at the N termini.

expressed, and monitored for their localizations

expressed, and monitored for their localizations. The results
showed that, like myc-VSP9B10A (Fig. 2A and B), they all failed to
be expressed on the membrane surface (see Fig. S2 in the supplemental material). A plausible explanation for it would be that
there is no targeting signal at the N terminus of VSP, while the
translocalization of VSP is blocked by tagging the N terminus of
VSP with 3 myc.
There are 73 vsp genes in the G. lamblia WB isolate. With the
establishment of motif 1 and motif 2 as the two essential and
sufficient structural elements in a VSP molecule for translocalization, we used them to screen the GiardiaDB and were able to identify a total of 73 vsp genes in the G. lamblia WB isolate.

VSPs all carry motif 1 and motif 2 virtually identical to those presented among the 11 experimentally verified VSPs in Fig. 4A
and are thus most likely the bona fide VSPs in Giardia.

An examination of the sequences in the N domains of the 73 proteins indicated vast diversities, suggesting that they are 73 distinctive proteins without a common N-terminal targeting signal. The
molecular masses of the 73 VSPs range from 228.40 kDa for
VSP-67 to 10.49 kDa for VSP-117, which consists of primarily
only motifs 1 and 2 (see Table S1 in the supplemental material).
Among the 73 VSPs, only 5 do not have the C-terminal CRGKA
and 3 have modified CRGKA. The amino acid sequences from the
beginning of motif 1 to the C-terminal end of motif 2 were aligned
among the 73 VSPs (Fig. 7). A dendrogram of the 73 sequences
was drawn according to the pairwise similarity of the sequences
using Clustal W (see Fig. S3 in the supplemental material). Based
on the dendrogram, the 73 VSP protein sequences could fall into
three subgroups I, II, and III (Fig. 7; see also Fig. S3).

Most of the
VSPs are classified to subgroup I, which includes all the VSPs
experimentally verified thus far. Although those in subgroups II
and III remain to be experimentally verified, chances are that they
are also bona fide VSPs due to the extreme sequence similarities
among all 73 VSPs (Fig. 7). These 73 VSPs may thus represent the
core VSPs in Giardia. Further studies will be required to address
whether minor structural modifications of motifs 1 and 2 will be
possible without affecting the membrane localization of a VSP.

addition to the zinc finger

In addition to the zinc finger, there are also other conserved
regions in motif 1, whose potential significance remains unknown
at the present time. There are 3 other proteins identified in the
GiardiaDB that contain only motif 1 but not motif 2. They have all
been annotated as high cysteine membrane proteins, even though
the membrane location has not yet been experimentally verified.
The highly conserved hydrophobic region of 23 amino acid residues in the middle of motif 2 constitutes most likely the transmembrane domain anchoring the VSP to cell membrane.

The GiardiaDB also shows 21 additional proteins that carry motif 2 at their C termini without motif 1. They include several previously
annotated VSPs, one hypothetical protein, and 3 other proteins
designated high cysteine membrane proteins. By our current experimental proof that both motif 1 and motif 2 are required for
localizing VSPs to the cell membrane surface, none of the abovementioned proteins could be classified as VSPs.
Our experimental results indicated also that the C-terminal
CRGKA is not required for VSP expression on the Giardia cell
membrane. This is in contrast to a previous report (29), which
showed that, during encystation of Giardia isolate GS, removal of
C-terminal CRGKA from a chimera reporter protein containing
the 43-amino-acid C terminus of VSPH7 resulted in failure of
translocalizing the protein to the plasma membrane.

The chimera protein apparently contains only motif 2 but not motif 1 identified in VSPs by us. It is thus not clear how the reporter
translocalized to the plasma membrane at all. Since the chimera
contains also the N-terminal leader sequence of a cell wall protein
1 and a Toxoplasma gondii SAG1 exodomain, its membrane expression could be by a different mechanism from that of VSP

A subsequent investigation indicating that a CRGKA
tail-deleted mutation and a point mutation of the Cys residue orthe Arg residue in the CRGKA tail of VSPH7 could still localize the
latter to the cell membrane of the trophozoites of Giardia isolate
GS appears to be in good agreement with our current finding (30,
31). However, since most of the newly identified 73 VSPs carry
CRGKA at their C termini (Fig. 7), this pentapeptide should remain a hallmark of VSPs, performing a certain function not yet

Our study also indicates that the N termini of VSPs cannot be
blocked by c-myc, or the translocalization of VSP will be inhibited.
But there does not appear to be an N-terminal-specific sequence
requirement for the signaling function.

Among the other anaerobic protozoan parasites, Entamoeba
histolytica has numerous receptor serine/threonine kinase proteins carrying motifs with 50% sequence similarities to motif 1
near their N termini. But they do not possess motif 2. These proteins were postulated to localize to the cell membrane surface

function in cell signaling (32). About 40 proteins in Trichomonas vaginalis have been found to have motifs at their C termini,
with sequences similar to that of motif 2, though motif 1 is absent.
Among these proteins, 4 are the surface immunogen P270-related
proteins, whereas 18 were detected in the proteomic analysis of the
membrane surface proteins from T. vaginalis (33). Neither E. histolytica nor T. vaginalis possesses proteins containing both motif


Across intra-mammalian stages of the liver f luke Fasciola hepatica: a proteomic study
Across intra-mammalian stages of the liver f luke Fasciola hepatica: a proteomic study

The presence of these two motifs in Giardia VSPs may thus
constitute a specific structural feature.
Another protozoan parasite known to possess a single species
of variant surface glycoprotein (VSG) covering the cell membrane
surface is the African trypanosome Trypanosoma brucei. The
bloodstream form of the T. brucei cell expresses a single species of
VSG on the membrane surface at any given time but is regularly
replaced by another VSG with an estimated frequency of up to
103 per cell per generation in an apparent effort of evading the
host immune response (34, 35). The VSGs do not contain either
motif 1 or motif 2 and are anchored to the cell membrane by a
specific glycolipid anchor (35). The expression of a vsg gene in
T. brucei requires a duplication of the gene, which is followed by a
translocalization of the duplicated gene to the expression site at
subtelomeric locations (36). Most of the vsp genes in Giardia are,
however, located in chromosome-internal positions, and a subtelomeric localization is apparently not required for their expression
(3). The mechanism of genetic regulation of VSP expression in
Giardia has been recently investigated by us. A functional microRNA (miRNA) machinery is apparently present in this organism (37). Among the 6 miRNAs we have identified thus far, 5 of
them, miR2, miR4, miR5, miR6, and miR10, have found their
corresponding target sites at the 3= ends of many tentative vsp
genes (16, 37–39). Translational repression by these miRNAs on
the expression of the encoded proteins carrying the corresponding
target sites on the mRNAs was verified by extensive experiments
(16, 37–39). With the newly verified spectrum of 73 VSPs made
available in our present study, we repeated the search for the target
sites of the 5 known miRNAs among the 73 vsp genes (16, 37–39).
The results, presented in Table S1 in the supplemental material,
indicate that most of the genes (88%) possess at least one target
site for one of the 5 known miRNAs. Since we have not yet exhausted the pool of miRNAs in Giardia, we anticipate that more
miRNAs will be found involved in regulating the expression of the
73 vsp genes. Each of the genes may thus turn out possessing multiple targeting sites for multiple miRNAs. The expression of most
of the vsp genes is thus most likely repressed. The mechanism involved in allowing the expression of only a single VSP species
among the 73 at a given time may thus include regulation of the
expression of various miRNAs, which could be also the basis for
periodic variation of the expression of VSP. Further studies will be
required for exploring this fascinating biological phenomenon.
Cloning of 3 myc-tagged vsp genes. The entire coding region of each
chosen putative vsp gene was PCR amplified from Giardia genomic DNA.
The product was cloned into pGEM-T Easy (Promega), sequenced, and
subcloned into the pNlop4 vector, a derivative of the pNlop3-GTetR vector kindly provided by Zac Cande of UC Berkeley (40). For expressing
N-terminal 3 myc-tagged VSPs, the pNlop4 vector was modified to
introduce each vsp gene downstream from a 3 myc epitope. For expressing C-terminal 3 myc-tagged VSPs, the pNlop4 vector was modified to
introduce the vsp gene and the 3 myc epitope upstream from the TAA
stop codon. Figure S4 in the supplemental material shows the schematic
representation of the N-terminal and C-terminal 3 myc-tagged vsp constructs used in this study. The pNlop4 vector that carries no tag was used
for cloning individual vsp genes without any tag.
Cell culture, transfection, and selection. Giardia lamblia (WB clone
C6, ATCC 50803) trophozoites were grown anaerobically in plastic culture tubes at 37°C in the modified TYI-S-33 medium supplemented with
antibiotics as described (41). Transfections of Giardia trophozoites were
carried out using electroporation. Cells at mid- to late-logarithmic phase
were harvested by chilling the culture tubes on ice for 10 min and collected
by a brief centrifugation (1,000  g at 4°C for 10 min). The cells were
washed twice in phosphate-buffered saline (PBS) and once in electroporation buffer (cytomix buffer; 10 mM K2HPO4-KH2PO4 [pH 7.6], 25 mM
HEPES free acid, 120 mM KCl, 0.15 mM CaCl2, 2 mM EGTA, 5 mM
MgCl2, 2 mM ATP, 4 mM glutathione) and then suspended to a final
concentration of 2.5  107 cells/ml. An aliquot of the concentrated cell
suspension (400 l, containing 107 cells) was transferred to a 0.2-cm-gap
electroporation cuvette (Bio-Rad) and placed on ice. A sample of 50 g
plasmid DNA was added to the cell suspension. The cells were immediately subjected to electroporation using a Bio-Rad Gene Pulser Xcell (voltage, 450 V; capacitance, 500 mF; resistance, ). The electroporated cells
were incubated on ice for 10 min, added to prewarmed culture medium,
and incubated at 37°C. For selection, 200 g/ml G418 was added to the
medium 16 h after transfection. The selected cells were incubated with
5 g/ml of tetracycline at 37°C for 16 h to induce expression of the cloned
Western blotting. The Mem-PER eukaryotic membrane protein extraction reagent kit (Thermo Scientific) was used to extract proteins from
the cell lysate. The separated aqueous and organic phases were each purified and concentrated using the Pierce SDS-PAGE sample prep kit
(Thermo Scientific). The concentration of protein in each sample was
quantified using the Bradford method (Bio-Rad). For SDS-PAGE separation, 25 g of protein from each sample was used. The fractionated proteins were then transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad) using the Trans-Blot SD semidry transfer cell (Bio-Rad).
The blot was used for detection with the anti-c-myc–horseradish peroxidase (HRP) antibody (Invitrogen) or the monoclonal antibodies for specific VSPs. The relative intensities of the stained bands were monitored by
densitometer tracing for a quantitative estimation of the distribution of
the protein between aqueous and organic phases.
Immunofluorescence assay. For detecting the 3 myc-tagged VSP
expression, the harvested Giardia cells were resuspended in 200 l of
modified TYI-S-33 culture medium, placed on a poly-L-lysine-coated
coverslip (BD Biosciences), and incubated at 37°C for 30 min to allow the
cells to adhere.