Dramatic reorganisation of Trichomonas endomembranes during amoebal transformation: A possible role for G-proteins

University of Cambridge, Cambridge, England, United Kingdom
Molecular and Biochemical Parasitology (Impact Factor: 1.79). 08/2006; 148(1):99-102. DOI: 10.1016/j.molbiopara.2006.02.022
Source: PubMed


Available from: Mark Field
Molecular & Biochemical Parasitology 148 (2006) 99–102
Short communication
Dramatic reorganisation of Trichomonas endomembranes during amoebal
transformation: A possible role for G-proteins
Kalpana Lal
, Christophe J. Noel
, Mark C. Field
, David Goulding
, Robert P. Hirt
Department of Zoology, The Natural History Museum, South Kensington, London SW7 5BD, UK
The Devonshire Building, School of Biology and Psychology, The University of Newcastle upon Tyne, NE1 7RU, UK
The Molteno Building, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK
The 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-
ally transmitted pathogen and is thought to represent a significant
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
upon binding to vaginal epithelial cells or to extracellular matrix
(ECM) proteins and can be induced in vitro upon binding to
ECM components laminin and fibronectin [3]. This process is
believed to involve specific cell signalling events [4]. Membrane
trafficking is also a key to Trichomonas pathogenesis; for exam-
ple perforin secretion leads to host cell lysis [5]. 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-
ficking and cell signalling in most organisms [6] and so we chose
to investigate the roles of a Trichomonas G-protein. We have
previously shown that the T. vaginalis G-protein alpha subunit
TvG402 locates at endomembranes in trophozoites [7].
Abbreviations: BSA, bovine serum albumin; ConA, concanavalin A; DAPI,
,6-diamidino-2-phenylindole dihydrochloride; DIC, differential interference
contrast; ECM, extracellular matrix; ER, endoplasmic reticulum; IFA, indirect
immunofluorescence analysis; TvG402, 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).
Present address: Infection and Immunity Section, Department of Biological
Sciences, Sir Alexander Fleming Building, Imperial College, South Kensington,
London SW7 2AZ, UK
Indirect immunofluorescence analysis (IFA) labelling of
TvG402 together with a nuclear stain (DAPI) shows TvG402
to localise to posterior perinuclear vesicular structures (up to
2 m in diameter) in Trichomonas trophozoite cells (Fig. 1A–D)
[7]. 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 [8] and we show ConA to partially co-localise
with TvG402 (Fig. 1E–H). ConA is likely to represent a highly
specific probe for Trichomonas glycoproteins as only two bands
were detected when a Trichomonas total cell extract was probed
with ConA by Western blot (Supplementary Fig. S1). ER tracker
labels the ER in many cell types [9,10] and in Trichomonas cells
appears to highlight several structures, including a large oval
structure similar to the nuclear membrane in size and location
with respect to TvG402. This is consistent with ER labelling.
However, TvG402 localisation is clearly fully distinct to ER
tracker indicating an absence of TvG402 at the ER (Fig. 1I–L).
A partial co-localisation with ConA (Fig. 1E) suggests that
TvG402 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, TvG402 organisation appears dis-
tinct from Golgi complex morphology as in Trichomonas the
Golgi complex is a single copy organelle located anterior to the
nucleus [11], whilst TvG402 labels structures posterior in the
cell (Fig. 1A) [7]. This leads us to suggest that TvG402 locates
at endosome/lysosomes. Additionally, TvG402 labelled vesi-
cles share morphological similarities with Trichomonas endo-
some/lysosomes, which can grow very large (up to 2 min
diameter) and are preferentially positioned posterior within the
cell [12]. Immunoelectron microscopy shows Trichomonas to
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100 K. Lal et al. / Molecular & Biochemical Parasitology 148 (2006) 99–102
Fig. 1. Cellular localization of TvG402 compared to different markers. TvG402 at posterior vesicles with partial co localisation with ConA binding but distinct
to ER tracker. TvG402 vesicles are frequently linked to endocytosed dextran but not ConA. Ultrastructure shows heterogeneous location of TvG402 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-TvG402 (1/5000) and DAPI (A–D) or ConA (E-H). Anti-TvG402 antibody (M006) was detected with anti-rabbit Alexa 488. Live Trichomonas
cells were incubated with ER tracker for 30 min and processed for IFA (I–L). Trichomonas endocytosis was monitored after incubation with dextran-texas red for
12 min (M–P) or ConA-biotin (Q–T) for 2 min and chased in buffer for 10 min 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 TvG402 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 TvG402 labelling with individual co-stains are shown in the first column. Panel (A) shows TvG402 at posterior perinuclear vesicular
structures. TvG402 and ConA labelling (panel E) shows a partial co-localisation. In panel (I), the overlay image of TvG402 and ER tracker clearly shows distinct
labelling. Dextran labelled vesicles often appear inside TvG402 (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-TvG402-protein A gold, show TvG402 at heterogeneous
vesicles. Multivesicular bodies-like compartments have membrane bound lumenal vesicles and TvG402 location includes the cytoplasmic face of the outer limiting
membrane of such compartments. TvG402 is also found at unstructured membranes in the cytosol. The bars represent 500 nm.
have multivesicular body-like compartments, i.e. vesicles con-
taining lumenal vesicles. TvG402 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).
To further investigate the relationship of TvG402 to endocy-
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. [13]. Dextran-labelled endocytic vesicles, less than 1 min
diameter, frequently appear within TvG402 stained structures
(Figs. 1M–P, Fig. S2 and movie 1) [7]. Thus a subset of TvG402
positive vesicles are linked to endocytosis. This staining pattern
is consistent with dextran labelling luminal vesicles of multivesi-
cluar bodies-like compartments whilst TvG402 labels the outer
membranes, as suggested from the immunoelectron microscopy
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
TvG402 (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 TvG402 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-
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K. Lal et al. / Molecular & Biochemical Parasitology 148 (2006) 99–102 101
Fig. 2. TvG402 location is dramatically altered with trophozoite to amoeba transformation. The cellular location of TvG402 is compared in trophozoite cells
(A and B) to amoeba after incubation with ECM proteins for 20 or 30 min. Within 20 min of contact with ECM proteins Trichomonas start to transform into early
amoeboid forms (C and D). After 30 min, 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 TvG402 (green) and DAPI stained the nucleus (blue). Panels B, D and F show the respective corresponding DIC images. TvG402 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 TvG402 is found
at the cell periphery in reduced numbers and often in pseudopodia (E and F). TvG402 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
Binding to ECM proteins fibronectin and laminin induces dif-
ferentiation of Trichomonas trophozoites into an amoebae form
[3]. After 20 min of contact with ECM proteins the cellular mor-
phology of T. vaginalis clearly reflects the differentiation process
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 TvG402
labelled structures differentiate into well-defined vesicles, which
proliferate in number (from 3 to >10 per cell) and dramati-
cally occupy most of the cell volume (Fig. 2C and Movie 2).
The TvG402 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, TvG402 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
TvG402 mRNA in amoebae bound to ECM proteins compared
to trophozoites (Fig. S3 [19]). It is also possible that the fraction
of TvG402 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
together with membrane reorganisation (Fig. 2C). Therefore the
increased TvG402 signal from trophozoite to amoeba detected
by immunofluorescence is likely due to the combined effect of
a burst in TvG402 protein synthesis and the possible reloca-
tion of the original pool of protein together with endomembrane
reorganisation. After 30 min of contact with ECM proteins, in
Trichomonas “late” amoebae, TvG402 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
involving an increase in TvG402-labelled vesicle numbers and
a later phase where these are reduced. At present the significance
of the realignment of TvG402-labelled membranes is not clear
but the relocation of vesicles towards the cell surface suggests a
role in secretion. In better-characterised systems G-proteins have
a well-established role at multivesicluar bodies in the secretion
of exosomes [15,16].AsTrichomonas stores HIV particles at
multivesicluar bodies-like compartements [17] reminiscent of
TvG402-positive compartments, it is possible that TvG402
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102 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 TvG402 and its
associated organelles is clearly of importance.
This work was funded by a Wellcome Trust University Award
to RPH (grant #060068) and a European Union Marie Curie Indi-
vidual Fellowship to CN (contract #HPMF-CT-2002-02071).
This work also benefited from program grant funding from the
Wellcome Trust to MCF.
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at doi:10.1016/j.molbiopara.2006.02.022.
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    • "The free-swimming ovoid cells, which resemble the familiar image of a flagellated protozoan, transform into an amoeboid form (Fig. 1) upon contact with the urogenital tract. This process commences more or less immediately upon contact with host tissue and the transformation of a cell takes only minutes to complete (Lal et al., 2006). The parasite's morphogenesis entails at least two distinct but simultaneous processes: (i) the dramatic shape transition and (ii) the adherence to host cells. "
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