Molecular & Biochemical Parasitology 148 (2006) 99–102
Dramatic reorganisation of Trichomonas endomembranes during amoebal
transformation: A possible role for G-proteins?
Kalpana Lala,1, Christophe J. Noelb, Mark C. Fieldc, David Gouldingd, Robert P. Hirtb,∗
aDepartment of Zoology, The Natural History Museum, South Kensington, London SW7 5BD, UK
bThe Devonshire Building, School of Biology and Psychology, The University of Newcastle upon Tyne, NE1 7RU, UK
cThe Molteno Building, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK
dThe Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK
Received 21 December 2005; received in revised form 21 February 2006; accepted 28 February 2006
Available online 31 March 2006
Keywords: Trichomonas vaginalis; Amoeba transformation; G-protein; Membrane trafficking; Intracellular localisation; Multivesicular bodies; Secretion
Trichomonas vaginalis is the most common non-viral sexu-
risk factor for HIV infection [1,2]. Trichomonas transformation
from a free-swimming trophozoite to an adherent amoeba is
crucial to parasite establishment in the host vagina and subse-
quent pathogenesis [1–4]. Amoebal transformation takes place
(ECM) proteins and can be induced in vitro upon binding to
ECM components laminin and fibronectin . This process is
trafficking is also a key to Trichomonas pathogenesis; for exam-
ple perforin secretion leads to host cell lysis . Despite the
potential importance of membrane trafficking and signalling
to these clinical aspects of Trichomonas, little is known of
the molecular machinery orchestrating these processes. Het-
erotrimeric G-proteins (G-proteins) are key to membrane traf-
to investigate the roles of a Trichomonas G-protein. We have
previously shown that the T. vaginalis G-protein alpha subunit
TvG?402 locates at endomembranes in trophozoites .
Abbreviations: BSA, bovine serum albumin; ConA, concanavalin A; DAPI,
4?,6-diamidino-2-phenylindole dihydrochloride; DIC, differential interference
contrast; ECM, extracellular matrix; ER, endoplasmic reticulum; IFA, indirect
immunofluorescence analysis; TvG?402, Trichomonas G-protein alpha subunit
?Supplementary data are associated with this article.
∗Corresponding author. Tel.: +44 191 246 4805; fax: +44 191 246 4998.
E-mail address: R.P.Hirt@ncl.ac.uk (R.P. Hirt).
1Present address: Infection and Immunity Section, Department of Biological
London SW7 2AZ, UK
Indirect immunofluorescence analysis (IFA) labelling of
to localise to posterior perinuclear vesicular structures (up to
. There are few Trichomonas intracellular markers charac-
terised but the mannose-specific lectin Concanavalin A (ConA)
preferentially binds glycoproteins at the ER, Golgi and endo-
some/lysosomes  and we show ConA to partially co-localise
specific probe for Trichomonas glycoproteins as only two bands
were detected when a Trichomonas total cell extract was probed
appears to highlight several structures, including a large oval
structure similar to the nuclear membrane in size and location
with respect to TvG?402. This is consistent with ER labelling.
However, TvG?402 localisation is clearly fully distinct to ER
A partial co-localisation with ConA (Fig. 1E) suggests that
TvG?402 locates at either the ER, Golgi or lysosomes; the for-
mer compartment can be eliminated due to a lack of overlap
with ER tracker. Further, TvG?402 organisation appears dis-
tinct from Golgi complex morphology as in Trichomonas the
Golgi complex is a single copy organelle located anterior to the
nucleus , whilst TvG?402 labels structures posterior in the
at endosome/lysosomes. Additionally, TvG?402 labelled vesi-
cles share morphological similarities with Trichomonas endo-
some/lysosomes, which can grow very large (up to ∼2?m in
diameter) and are preferentially positioned posterior within the
cell . Immunoelectron microscopy shows Trichomonas to
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K. Lal et al. / Molecular & Biochemical Parasitology 148 (2006) 99–102
Fig. 1. Cellular localization of TvG?402 compared to different markers. TvG?402 at posterior vesicles with partial co localisation with ConA binding but distinct
to ER tracker. TvG?402 vesicles are frequently linked to endocytosed dextran but not ConA. Ultrastructure shows heterogeneous location of TvG?402 including the
cytoplasmic face of multivesicular body-like compartments. Trichomonas cells were fixed with ethanol or parformaldehyde and permeablised with Triton X-100 and
labelled with anti-TvG?402 (1/5000) and DAPI (A–D) or ConA (E-H). Anti-TvG?402 antibody (M006) was detected with anti-rabbit Alexa 488. Live Trichomonas
cells were incubated with ER tracker for 30min and processed for IFA (I–L). Trichomonas endocytosis was monitored after incubation with dextran-texas red for
12min (M–P) or ConA-biotin (Q–T) for 2min and chased in buffer for 10min and processed for IFA. ConA-biotin was detected with streptavidin texas red. Images
(A–D) and (I–L) were captured with an epifluorescent microscope whilst the remainder were single section confocal images. Images in each row represent one cell
and TvG?402 staining is shown in green in the first and second columns. Various co-stains indicated to the left of each row are shown in blue or red in the third
column. The overlays of TvG?402 labelling with individual co-stains are shown in the first column. Panel (A) shows TvG?402 at posterior perinuclear vesicular
structures. TvG?402 and ConA labelling (panel E) shows a partial co-localisation. In panel (I), the overlay image of TvG?402 and ER tracker clearly shows distinct
labelling. Dextran labelled vesicles often appear inside TvG?402 (M–P) outlined vesicles whilst ConA labelled vesicles do not (Q–T). The bars represent 2?m.
In panels (U and V), immuno electron microscopy images of Trichomonas cells labelled with anti-TvG?402-protein A gold, show TvG?402 at heterogeneous
vesicles. Multivesicular bodies-like compartments have membrane bound lumenal vesicles and TvG?402 location includes the cytoplasmic face of the outer limiting
membrane of such compartments. TvG?402 is also found at unstructured membranes in the cytosol. The bars represent 500nm.
have multivesicular body-like compartments, i.e. vesicles con-
taining lumenal vesicles. TvG?402 localisation includes the
cytoplasmic face of the outer limiting membrane of multivesi-
cluar bodies-like compartements and also at unstructured mem-
branes (Fig. 1U and V).
tosis we tracked endocytic material using the probes dextran or
ConA. The endocytic marker fluorescent dextran (Mwt 10,000
daltons) is taken into many cell types by a fluid-phase route –
e.g. . Dextran-labelled endocytic vesicles, less than 1?m in
diameter, frequently appear within TvG?402 stained structures
positive vesicles are linked to endocytosis. This staining pattern
data (Fig. 1U and V).
Alternatively, ConA binds glycoproteins on the surface of
live Trichomonas cells and allows monitoring of endocytosis
of ConA-labelled glycoproteins. In contrast to dextran uptake,
ConA-labelled endosomes do not appear to be associated with
TvG?402 (Fig. 1Q–T). Also, the kinetics of dextran and ConA
uptake are different (Supplementary Fig. S2). Thus, dextran and
ConA appear to label distinct populations of vesicles. Also, not
all endocytic cargo taken into the cell is passively transported
within TvG?402 labelled vesicles. Thus the potential complex-
ity of Trichomonas endocytosis is also demonstrated here with
the active sorting of the contents of multivesicluar bodies-like
compartements. Further, the recent discovery of an extremely
large Rab GTPase gene family, encoding proteins key to mem-
K. Lal et al. / Molecular & Biochemical Parasitology 148 (2006) 99–102
Fig. 2. TvG?402 location is dramatically altered with trophozoite to amoeba transformation. The cellular location of TvG?402 is compared in trophozoite cells
(A and B) to amoeba after incubation with ECM proteins for 20 or 30min. Within 20min of contact with ECM proteins Trichomonas start to transform into early
amoeboid forms (C and D). After 30min, late amoebae cells show a reduced size and pseudopodia are often apparent (E and F). Trophozoites were fixed and adhered
to slides with poly-l-lysine whilst amoebae adhered via ECM proteins for indicated times and then fixed and processed for IFA. Panels A, C and E show overlay
images of TvG?402 (green) and DAPI stained the nucleus (blue). Panels B, D and F show the respective corresponding DIC images. TvG?402 labelled structures
change from a perinuclear posterior location in the trophozoite (A), to occupying most of the cell in the early amoeba (C). In the late amoeba TvG?402 is found
at the cell periphery in reduced numbers and often in pseudopodia (E and F). TvG?402 labelling appears brighter and more condensed at vesicles in the amoebal
stages. The bar represents 2?m.
brane trafficking in model systems, also strongly suggests an
exceedingly complex endomembrane system in Trichomonas
ferentiation of Trichomonas trophozoites into an amoebae form
as cells in transition show an irregular outline under differential
interference contrast (DIC) that is distinct from the trophozoite
(compare Fig. 2B and D), indicating remodelling of the cell
surface and overall cell shape. During this process TvG?402
proliferate in number (from ∼3 to >10 per cell) and dramati-
cally occupy most of the cell volume (Fig. 2C and Movie 2).
The TvG?402 labelled vesicles appear with a similar morphol-
ogy in terms of shape and size as in the trophozoite, suggesting
the labelling of endosomal/lysosomal compartments into early
differentiation. Strikingly, TvG?402 labelling appears brighter
and more distinct at vesicles during the differentiation to the
amoeba (comparing Fig. 2A and C and Movies 1 and 2). Semi-
quantitative RT-PCR analyses shows a dramatic increase in
to trophozoites (Fig. S3 ). It is also possible that the fraction
of TvG?402 in the trophozoite that is observed as diffuse stain-
ing by immunofluorescence (Fig. 2A) and likely at cytoplasmic
membranes by electron microscopy (Fig. 1U and V), may relo-
cate to vesicle membranes during the amoebal transformation
by immunofluorescence is likely due to the combined effect of
a burst in TvG?402 protein synthesis and the possible reloca-
tion of the original pool of protein together with endomembrane
reorganisation. After 30min of contact with ECM proteins, in
Trichomonas “late” amoebae, TvG?402 labelled vesicles often
relocate to a single focus towards the cell periphery, often at
pseudopodia (Fig. 2E and F).
The present data demonstrates dramatic changes occurring
in the Trichomonas endomembrane system that accompany
amoeba transformation following contact with ECM proteins.
Significantly these studies also highlight at least two develop-
mental phases in the amoeboid transformation, an early phase
but the relocation of vesicles towards the cell surface suggests a
a well-established role at multivesicluar bodies in the secretion
of exosomes [15,16]. As Trichomonas stores HIV particles at
multivesicluar bodies-like compartements  reminiscent of
TvG?402-positive compartments, it is possible that TvG?402
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K. Lal et al. / Molecular & Biochemical Parasitology 148 (2006) 99–102
plays an important role in the release of harboured HIV upon
contact to host tissue and contributes to the well established
increased risk of HIV infection for Trichomonas infected peo-
ple [1,17,18]. Further study of the functions of TvG?402 and its
associated organelles is clearly of importance.
vidual Fellowship to CN (contract #HPMF-CT-2002-02071).
This work also benefited from program grant funding from the
Wellcome Trust to MCF.
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