Microbes and Infection 14 (2012) 1411e1427

www.elsevier.com/locate/micinf

The effects of environmental factors on the virulence of Trichomonas

vaginalis
Elisa E. Figueroa-Angulo a, Francisco J. Rendón-Gandarilla a, Jonathan Puente-Rivera a,
Jaeson S. Calla-Choque a, Rosa E. Cárdenas-Guerra b, Jaime Ortega-López b,
Laura I. Quintas-Granados c, M. Elizbeth Alvarez-Sánchez c, Rossana Arroyo a,*
a
Departamento de Infectómica y Patoge´nesis Molecular, Centro de Investigación y de Estudios Avanzados del Instituto Polite´cnico Nacional (CINVESTAV-IPN),
Av. IPN # 2508, Col. San Pedro Zacatenco, Me´xico, D.F., CP 07360, Mexico
b
Departamento de Biotecnologı´a y Bioingenierı´a, Centro de Investigación y de Estudios Avanzados del Instituto Polite´cnico Nacional (CINVESTAV-IPN),
Av. IPN # 2508, Col. San Pedro Zacatenco, Me´xico, D.F., CP 07360, Mexico
c
Posgrado en Ciencias Genómicas, Universidad Autónoma de la Ciudad de Me´xico, plantel del Valle, San Lorenzo # 290, Col. Del Valle, Me´xico, D.F.,
CP 03100, Mexico
Received 9 February 2012; accepted 2 September 2012
Available online 25 September 2012

Abstract

This review focused on potential regulatory mechanisms of Trichomonas vaginalis virulence properties, cytoadherence, cytotoxicity,
phagocytosis, hemolysis, induction of apoptosis, and immune evasion in response to environmental factors of the human urogenital tract, iron,
zinc, and polyamines. Understanding the multifactorial nature of trichomonal pathogenesis and its regulation may help to unravel the survival
strategies of trichomonads and to implement prevention policies, opportune diagnosis, and alternative treatments for control of trichomoniasis.
Ó 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Keywords: Trichomonas vaginalis; Virulence; Iron; Cell contact; Zinc; Polyamines

1. Introduction rapidly clear the infection, suggesting that this differential

Human trichomoniasis is a sexually transmitted infection
caused by the protist parasite Trichomonas vaginalis. This
chronic infection is the most common non-viral sexually
transmitted disease worldwide. Infection often leads to vagi-
nitis in women or urethritis and prostatitis, in men. Tricho-
moniasis is associated with preterm delivery, low birth weight,
and increased infant mortality. This infection also predisposes
individuals to HIV/AIDS and cervical and prostatic cancers. T.
vaginalis can also be responsible for pneumonia, bronchitis,
and oral lesions in immunocompromised patients [1].
Trichomoniasis is a gender-related infection mainly
affecting women, whereas the majority of men are able to
* Corresponding author. Tel.: þ52 55 5747 3342; fax: þ52 55 5747 3377.
E-mail address: rarroyo@cinvestav.mx (R. Arroyo).
scenario may be related to differences in the urogenital
microenvironments affecting trichomonad pathobiology. T.
vaginalis encounters an iron-rich environment in the vagina
and a hostile, zinc-rich environment in the prostatic glands.
Zinc is a known antimicrobial chemical defense in humans;
men with <1.6 mM zinc in their prostatic secretions can suffer
chronic prostatitis due to T. vaginalis infection [2].
T. vaginalis responds to drastic environmental changes
(e.g., temperature, microflora, pH, iron, polyamines, zinc, host
immune responses, and other unknown factors), modulating
the expression of multiple genes, including those encoding
virulence factors, to maintain a chronic infection [1,3,4].
Additionally, because this parasite is microaerophilic, it
requires defense molecules to neutralize the effects of oxygen.
Genes encoding for superoxide dismutases, thioredoxin
reductases, peroxiredoxins, and rubrerythrins have been

1286-4579/$ - see front matter Ó 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.micinf.2012.09.004
1412 E.E. Figueroa-Angulo et al. / Microbes and Infection 14 (2012) 1411e1427

identified in the trichomonad genome [5]. To establish an
infection in humans, T. vaginalis binds and degrades the
vaginal mucous and specifically adheres to vaginal and
cervical epithelial cells (cytoadherence) [1]. This parasite can
also bind to prostatic cell lines in vitro and degrade them in
a contact-depend process as well [6]. However, T. vaginalis
does not undergo the same morphological changes with other
cell types as they do with vaginal epithelial cells (VECs) [6,7].
Upon contact with VECs, T. vaginalis undergoes a drastic
morphological shift, changing from the usual pear shape to an
ameboid form with an increase in the expression of adhesins
and a release of chemoattractant molecules which attracts
more parasites to the site of infection [7]. The morphological
transformation leads to the formation of areas where the
parasite and the target cell become tightly associated.
Membrane fusion is often observed during this process, sug-
gesting that signals can be exchanged between parasites and
host cells [4,8]. Parasite morphology is directly related to
cytoskeletal structure, and changes in parasite shape are
mediated by the redistribution of its molecular components.
However, these morphological changes were not observed
when trichomonads interacted with cervical or prostatic cells.
As revealed by scanning electron microscopy, the morphology
of T. vaginalis in contact with DU145 prostatic cells retains
a pear-like shape [6,7] (Fig. 1).
The participation of the cytoskeleton in pathogenicity has
also been highlighted by studies showing that anti-cytoskeletal
drugs can interfere with the cytopathological process. T. vag-
inalis possesses an actin cytoskeleton and several of its protein
components have already been characterized including a-
actinin, an actin-binding protein that actively participates in
the morphological transformation mediating the redistribution
of actin and two fimbrins, which belong to the actin-bundling
proteins and express in the presence of zinc [6,9]. T. vaginalis
a-actinin is evenly distributed throughout the cytoplasm when
the cell is pear-shaped. In ameboid parasites, a-actinin local-
izes only at the cell periphery, lining the pseudopodial
extensions of the transforming cell [9]. Here we will discuss
changes of T. vaginalis virulence as it relates to environmental
conditions. Due to space limitation, we emphasize mostly
recent review articles, as they include many original findings
contributing to the current state of knowledge.
2. Virulence mechanism of T. vaginalis
Although pathogenesis in T. vaginalis is not completely
understood, a successful colonization of the mucous
membranes and the maintenance of infection are likely
favored by the existence of several virulence mechanisms
including binding and degradation of mucosal and extracel-
lular matrix (ECM) components and the adherence to and
cytotoxicity of VECs, and cervical (HeLa) and prostatic cells
(DU145). Phagocytosis of vaginal microorganisms (bacteria,
viruses, yeast, etc.) and host cells (VECs, erythrocytes,
spermatozoa, etc.) [10e12] and the control and destruction of
cells comprising the immune system (e.g., neutrophils and
macrophages) by the induction of apoptosis are among the
multiple mechanisms involved in host immune evasion.
Other immune evasion strategies include the endocytosis
of host proteins and the degradation of complement
proteins, immunoglobulins, and other proteins, together with
surface molecular mimicry and masking with host proteins
[1,13].
2.1. Cytoadherence
Parasite cytoadherence is a prerequisite for establishing
and maintaining a chronic infection in human urogenital tract

Fig. 1. Morphological appearance of T. vaginalis in contact with DU145 prostatic cells at different time points. A. Trophozoites were incubated with DU145 cells
for a) 5 min, b) 30 min, c) 60 min, and d) 90 min. DU145 prostatic cells before interacting with T. vaginalis were used as a control (inset of panel a). The majority
of parasites attached to DU145 cells retain a pear-like shape with four flagella, an undulating membrane, and the axostyle. Some parasites showed a few pseu-
dopods (panel b and c). After 5 min of interaction, the lytic activity of the parasites is visible as seen by the disruption of the cell monolayer. This figure is part of
Fig. 4 taken from reference [6] with permission from the publisher.
 E.E. Figueroa-Angulo et al. / Microbes and Infection 14 (2012) 1411e1427
[1]. Cytoadherence is a complex process that involves not
only surface proteins “adhesins” and glycoconjugates but also
proteins in the cytoskeleton (consisting of microtubules and
microfilaments), the receptors for ECM proteins (laminin,
fibronectin, and collagen), and signal transduction and auto-
phagy processes [5,7,14e18]. Iron and cellular contact
positively regulate the levels of adherence by directly
increasing the adhesin synthesis [7,14,17e20]; but also
down-regulate the expression of several antigenic proteins
such as P270 [21].
Five T. vaginalis surface proteins that interact with the host
cell surface have been characterized as adhesins (AP120,
AP65, AP51, AP33, and AP23) [14,18]. Four of them are
metabolic enzymes [pyruvate:ferredoxin oxidoreductase
(PFO), malic enzyme, and a and b succinyl-CoA synthetase
subunits, respectively], suspected of having dual localization
inside trichomonads (hydrogenosomes and parasite surface).
Interestingly, the AP23 has not been identified yet. Could it be
another metabolic enzyme such as the triosephosphate isom-
erase recently reported [22]. These adhesins have been iden-
tified and characterized as multifunctional proteins that show
different functions depending on their localization regulated
by iron, acting as metabolic enzymes in hydrogenosomes and
the cytoplasm or as adhesins on the parasite surface. These
adhesins also participate in the molecular mimicry mecha-
nisms involved in immune evasion [1,13] and as hemoglobin
(Hb) and Heme receptors.
These hydrogenosomal enzymes lack enzymatic activity
when localized at the parasite surface, performing additional
tasks as virulence factors in adherence [17,18]. Although
somewhat controversial, an explanation for their relocalization
to the parasite surface through a hydrogenosomal autophagy-
linked process has been suggested [16,23] and shown at
least for one of them, the PFO/AP120 adhesin [17]. This
conclusion is supported by the recently identification of genes
encoding several of the conserved ATG proteins involved in
autophagy in the T. vaginalis genome. Further information can
be found in two reviews [23,24].
The identification of hydrogenosomal proteins as adhesins
has been controversial among other reasons due to: i) These
molecules are detected only in the hydrogenosomes by using
a different set of antibodies. ii) These molecules bind to the
surface of different cells. iii) These enzymes do not possess
transmembranal domains (TMD) that may explain its surface
localization. It is noteworthy to mention that this is not an
isolated phenomenon observed in this protist. Data have been
accumulated around this topic in the literature for many
different cells and parasites. The well known term of moon-
lighting proteins was originally branded to accommodate
multifunctional proteins that included surface proteins with
non-enzymatic function in spite of being enzymes of the
carbohydrate metabolism [25]. Now the multifunctionality of
these proteins is explained as part of functional divergence
after gene duplication that can result in two alternative
evolutionary fates: one copy acquires a novel function (neo-
functionalization), or each copy adopts part of the tasks of
their parental gene (subfunctionalization) [26].
1413
Cysteine proteinases (CPs) localized to the parasite surface,
such as TvCP30 [27e29] and TvCP62 [30], the surface lip-
ophosphoglycan (TvLPG) [31], and several ECM receptors,
are also involved in trichomonal cytoadherence. TvCP30
participates in cytoadherence, is immunogenic and is present
in vaginal secretions in patients with trichomoniasis, and is
active at the pH and temperatures found in the vagina during
infection. This CP degrades proteins of the vaginal milieu such
as collagen (Coll) IV, fibronectin (Fn), and Hb [27e29].
Other metabolic proteins with alternative non-enzymatic
function are glyceraldehyde-3-phosphate dehydrogenase
(TvGAPDH) and a-enolase (TvENO-1). These proteins are
also found on the T. vaginalis surface, but their sequences lack
TMD and signal peptides (SP). These enzymes exhibit ligand-
binding activity, a non-enzymatic function that may play an
important role in colonization and invasion, probably under
distinct host environments. The expression and surface local-
ization of TvGAPDH, a fibronectin-binding protein in T.
vaginalis, is also up-regulated by iron [13,32]. Surface-
localized TvENO-1 binds plasminogen, and its synthesis is
increased following trichomonad cellular contact with VECs
[13,33]. Both GAPDH and enolase are known to be multi-
functional proteins in other pathogens [13]. Thus, it will be
very interesting to identify other alternative functions for these
trichomonad proteins.
The search for surface proteins in the T. vaginalis genome
by sequence comparison with the surface molecules of
prokaryotic and eukaryotic pathogens to discover novel puta-
tive virulence factors has led to the identification of ten
different families of proteins that are considered to be strong
candidates for surface proteins. More information regarding
these putative surface proteins can be found in recent reviews
[16,24]. Several examples of these families are listed in
Table 1.
One of these families is the BspA-like proteins, which
present a specific type of leucine-rich repeat (TpLRR). Bacterial
BspAs are capable of mediating binding to host epithelial cells,
ECM proteins and cell aggregation. This trichomonad multi-
gene family, with 911 hypothetical members, has 178 BspA-like
proteins with TMD, consistent with a binding function on the
cell surface [16,24]. Considering the bacterial origin of this
protein and the permanent contact between T. vaginalis and
vaginal bacteria, it has been suggested that the acquisition of
these genes may have been due to a lateral transfer mechanism.
In the T. vaginalis genome, 152 cases of possible prokar-
yoteeeukaryote lateral gene transfer have been described [5].
The differences among the members of this family lie in the
number of TpLRRs (ranging from three to over 30 repeats), the
presence of distinct repetitive sequences, and differences in the
cytoplasmic tail.
Data showed by Noël et al., 2010 strongly suggest that the
TvBspA proteins play diverse and pivotal roles in T. vaginalis
pathobiology by contributing to the invasion and long term
infections of the human urogenital tract. TvBspA-like proteins
are strong candidate surface proteins mediating interaction with
various mucosal landmarks including the mucus; VEC, urethra
epithelial cells and other host cells; ECM proteins and vaginal
1414 E.E. Figueroa-Angulo et al. / Microbes and Infection 14 (2012) 1411e1427

Table 1
Trichomonas vaginalis virulence factors: virulence mechanisms, environmental signals, and type of regulation.
Protein Virulence factor Virulence property Regulationa Regulation level Reference
Iron Contact PA Zinc
AP23 Adhesin Cytoadherence þ ND ND þ Transcriptional [14,19]
Translational
AP33 Adhesin Cytoadherence þ þ ND þ Transcriptional [6,14,19,20]
a-subunit of succinyl Translational
Co A synthetase
AP51 Adhesin Cytoadherence þ þ ND þ Transcriptional [14,19,20]
b-subunit of succinyl Translational
Co A synthetase
AP65-1 Adhesin Cytoadherence þ þ ND þ Transcriptionalb [6,14,19,20,69]
Malic enzyme Translational
AP120 Adhesin Cytoadherence þ ND ND ND Transcriptional [14,17,18]
pyruvate:ferredoxin Post-translationalc
oxidoreductase
BspA-like Adhesin Cytoadherence þ/ þ ND ND Transcriptional [16,24]
Host immune evasion
Calpain-like CP Proteases ND ND ND ND ND ND [16]
Cell-detaching Glycoprotein Cytolytic activity ND þ ND ND ND [40]
factor (CDF) 200 kDa
CP (30 kDa) Proteinase Cytoskeletal disruption ND þ ND ND ND [9]
GP63-like Metalloproteinase Host protein degradation ND þ ND ND Transcriptional [5,37]
Lectins HIV endocytosis Endocytosis ND ND ND ND ND [16]
M17-like Adhesin Adherence ND ND ND ND ND [16,24]
P230 Membrane molecule Phenotypic variation ND ND ND ND ND [1]
P270 Membrane molecule Phenotypic variation e ND ND ND Post-translationald [1,16,21]
Phospholipase A2 Lipase Cytotoxicity ND ND ND ND ND [41]
Pmp Adhesin Cytoadherence ND ND ND ND Transcriptional and [16,24,54]
Host immune evasion post-transcriptional
Pore-forming Transmembrane protein Cytolytic activity ND ND ND ND Transcriptional [9]
proteins (PFPs)
SAPLIP Saposin-like proteins Cytolytic activity ND ND ND ND ND [24]
(Trichopores)
Serine protease Proteases ND ND ND ND ND ND [16]
TVCP4 Surface CP Hemolysis þ ND ND ND Post-transcriptionale [77]
TvCP12 Surface CP Cytotoxicity e ND ND ND Post-transcriptionale [60,63]
TvCP30 Surface CP Cytoadherence e ND ND ND ND [27e29]
Protein degradation
TvCP39 Surface CP Cytotoxicity e ND þ e Post-transcriptionalf [6,45,63]
Igs degradation Post-translationalc,g
TvCP62 Secreted CP Cytoadherence ND ND ND ND ND [30]
TvCP65 Surface CP Cytotoxicity e ND þ e Post-transcriptionalf [3,42,43]
CP2, CP3, CP4 Secreted CPs Apoptosis e ND ND ND ND [52]
and CPT
TvENO-1 Plasminogen receptor Cytoadherence ND þ ND ND Transcriptional [13,33]
Enolase
TVF Cytolytic effector 250 kDa Cytolytic activity ND þ ND ND ND [39]
TvGAPDH Fn-binding protein Cytoadherence þ ND ND ND Transcriptional [13,32]
Glyceraldehyde-3-phosphate
dehydrogenase
TvLEGU-1 Surface CP Cytoadherence þ ND ND ND Transcriptional [60]
Post-translationalc,d
TvLIP Triacylglycerol lipase Hemolysis þ þ ND ND ND [51]
TvLPG Lipophosphoglycans Cytoadherence ND þ ND ND Post-translationalc [5,31,85]
cytotoxicity
VSP-like Adhesin Cytoadherence ND ND ND ND ND [16,24]
a
Regulation by environmental factors such as iron, cell contact (Cell), polyamines (PA), and zinc.
b
Iron-responsive promoter.
c
Glycosylation.
d
Phosphorylation.
e
IRE/IRP-like system mediated by RNAeProtein interactions.
f
Regulation mediated by RNAeProtein interaction between the mature eIF 5A and ERE hairpin.
g
Interaction with cystatins. ND: No determined; (þ) up-regulated; () down-regulated.
 E.E. Figueroa-Angulo et al. / Microbes and Infection 14 (2012) 1411e1427
microflora or cellecell adhesion during parasites swarming.
TvBspA could also mediate endocytosis of different host
proteins and viruses, as well as underpin phagocytosis of
bacteria and various host cells. Finally, TvBspA proteins could
orchestrate the modulation of the innate immune system
through TLRs signaling during infection and mediate immune
evasion through differential expression [34].
The T. vaginalis dense glycocalyx corresponds to the outer
layer on its cell membrane formed by different carbohydrate-
associated molecules, such as lipophosphoglycan (LPG)-like,
glycoproteins, and glycolipids. The TvLPG-like is one of the
most abundant components of the T. vaginalis glycocalyx and
is an important virulence factor that appears to mediate
parasite-host cell interaction to VECs via binding to human
galectin-1 [31,35]. The TvLPG-like glycoconjugate is now
named T. vaginalis lipoglycan (TvLG) that differs markedly
from Leishmania LPG and Entameba lip-
opeptidophosphoglycan (LPPG) and is devoid of phospho-
saccharide repeats. TvLG is composed of a polyrhamnose
backbone with branches of Xylose and poly-N-acetyllactos-
amine and sometimes lacto-N-biose (poly-LacNAc/LNB). The
poly-LacNAc side chains have been shown to be involved in
parasite:host cell interaction [36].
T. vaginalis mutant cells deficient in TvLPG-like glyco-
sylation showed reduced adherence and cytotoxicity to human
cervical cells. As the morphological transformation of the
ovoid trophozoites into the ameboid form is triggered
following binding to VECs and ECM [7], the binding of
TvLPG-like to human galectin-1 may be important in this
parasite’s morphological changes [24]. Additionally, genes
that encode enzymes involved in TvLG synthesis have been
identified in the trichomonad genome. TvLG is rich in rham-
nose, a deoxysugar absent in humans, making its biosynthetic
pathway an attractive drug target [5,36].
Another member of the putative surface proteins is the
GP63 family. There are 48 members of the GP63 protease
family, 37 of which are predicted to be transmembrane
proteins that are possibly involved in hosteparasite interac-
tions during infection. Ma et al. (2011) performed a prelimi-
nary study on the functions of GP63 in T. vaginalis (TvGP63),
demonstrating that the cell-surface localization of one highly
expressed member of the TvGP63 family in two trichomonad
isolates is involved in trichomonal cytotoxicity but not in
cytoadherence [37]. It will be interesting to determine their
localization, functions, and the environmental conditions that
modulate their expression and possible participation in T.
vaginalis cytopathogenicity.
2.2. Cytotoxicity
In vitro studies show that trichomonal cytopathogenicity is
a multistep process that involves a contact-dependent process.
This mechanism starts with the parasite cytoadherence fol-
lowed by a cytotoxicity effect that involves a cascade of
events resulting in the cytolysis, phagocytosis, and disinte-
gration of cell monolayers. However, contact-independent
cytolytic mechanisms have been also demonstrated and
1415
attributed to parasite secreted molecules [10,38]. This
mechanism requires multiple molecules involved in cellular
damage, such as cytolytic effectors. TVF is a 250 kDa, cell-
free T. vaginalis culture factor that induces the progressive
rounding and clumping of target cells without cell lysis [39].
Cell-detaching factor (CDF) is a glycoprotein (200 kDa)
responsible for detachment and is released into the medium
by T. vaginalis in contact with epithelial cells [40]. Other
factors include porins [9], phospholipase A2 [41], and several
surface located CPs displaying down-regulated expression in
the presence of iron and having an affinity for the surface of
HeLa cells, e.g., TvCP12, TvCP39, and TvCP65. TvCP39
and TvCP65 are immunogenic in patients with trichomoni-
asis, found in vaginal secretions, active at the pH and
temperature found in the vagina during infection, degrade
proteins of the vaginal milieu such as Coll IV and
Fn, require polyamines for their expression, and their
expression is down-regulated by iron and zinc [3,6,42e46]
(unpublished).
2.3. Phagocytosis
Light and electron microscopy studies have helped to
demonstrate that T. vaginalis is a phagocytic cell that is able to
efficiently ingest and degrade Döderlein’s lactobacilli, vaginal
and cervical epithelial cells, leukocytes, erythrocytes, yeast,
spermatozoids, and prostatic cells [6,10e12,47]. Although the
mechanism of phagocytosis has not been fully elucidated, at
least two different pathways have been observed: a) the
pathway used by professional phagocytes in which pseudo-
podia are extended toward the target cell; and b) a sinking
process without membrane extension. It has been suggested
that different proteins are involved in these processes. Two
surface proteins have been described as putative adhesins for
erythrocytes [48], whereas a mannose receptor on the parasite
surface has been suggested for yeast phagocytosis. A rear-
rangement of and dramatic changes in the distribution of the
actin cytoskeleton have also been reported, which may facil-
itate the ameboid morphological transformation observed
during phagocytosis [12,47]. Thus, it has been suggested that
phagocytosis may be considered to be a virulence mechanism
in addition to a mechanism for the acquisition of iron, lipids,
nucleotides, and other nutrients.
Although the specific molecules that participate in
phagocytosis have not yet been identified, in silico analysis
showed that a considerable diversity of proteins comprising
the membrane-trafficking machinery appears to be encoded
by the T. vaginalis genome. Homologs of well-established
components of the vesicle-formation machinery, including
vesicle coats and GTPases, were identified along with diverse
elements of the vesicle-fusion machinery including SNAREs,
Rab GTPases, and SM proteins. Additionally, dynamin and
members of the endosomal sorting complex required for
transport (ESCRT) were also identified [5,23]. This infor-
mation will be very useful in directing future studies of the
connection between phagocytosis and virulence and in iden-
tifying the molecular complexes involved in it.
1416 E.E. Figueroa-Angulo et al. / Microbes and Infection 14 (2012) 1411e1427
2.4. Cell lysis
T. vaginalis employs a combination of strategies for target
cell disruption. Several commonly used mechanisms such as
hemolysis and the induction of apoptosis are described below:
2.4.1. Hemolysis
For trichomonads, erythrocytes are an important source of
nutrients such as lipids and iron; their lysis is contact-dependent
and may occur in vivo [48e50]. The level of beta-hemolytic
activity in fresh T. vaginalis isolates correlates with clinical
virulence. Among the molecules possibly involved in hemolysis
by T. vaginalis, surface CPs, pore-forming proteins, and
phospholipase-A-like proteins have been identified as cytolytic
factors [9,28,41,42,49]. In a functional assay using blood agar,
another triacylglycerol lipase (TvLIP) was identified in a cDNA
expression library that may be a hemolytic factor in T. vaginalis.
TvLIP expression is up-regulated by iron [51].
Moreover, several surface CPs play roles in hemolysis,
implying a contact-dependent mechanism [28,49]. A non-
secreted 30 kDa CP that has high specificity for spectrin is
thought to be the main effector responsible for the cytoskeletal
disruption associated with the cytolytic mechanisms of T.
vaginalis [9]. The degradation of spectrin occurs prior to the
lysis of erythrocytes.
Hemolysis is dependent on Ca2þ concentration, temperature
(37  C), and pH; an acidic pH (6.5) is required for the secretion
and activity of the pore-forming proteins (PFPs) [9]. Other
parasitic protists such as Entamoeba histolytica and Naegleria
fowleri produce PFPs (amebapores and naegleriapores,
respectively) that are secreted. These PFPs belong to
a conserved family of saposin-like proteins (SAPLIPs) that are
found in phylogenetically distant organisms (e.g., protists and
mammals). Members of the SAPLIP family have the most
diverse functions yet share a common feature: interaction with
lipids.
In T. vaginalis, twelve SAPLIP predicted genes
(TvSaplip1e12) have been identified by genomic analysis. All
of the features displayed by TvSaplips make the predicted
proteins of this new family, named the trichopores, good
candidates as effectors contributing to the cytolytic effects of
T. vaginalis. However, given the heterogeneous nature of
SAPLIP functions, it seems unlikely that all TvSaplips are
directly involved in the cytopathogenicity of this parasite. It is
well known that SAPLIPs can have functions other than
cytolytic activity in different biological systems [24].
2.4.2. Apoptosis
Using a vaginal epithelial cell culture system and MS
analysis showed that in vitro T. vaginalis causes cell detach-
ment followed by cell destruction as a result of apoptosis
induced by secreted CPs in the 30 kDa range (CP2, CP3, CP4
and CPT). The amounts and proteolytic activities of these CPs
are down-regulated by iron, as reflected by a reduced ability to
induce apoptosis [52]. This iron response is similar to that
observed for the trichomonal cytotoxicity in cervical cells
(HeLa cell monolayers) in which two other CPs are involved:
TvCP65 and TvCP39 also down-regulated by iron [42,43,45].
We recently reported that CPT corresponds to TvCP39,
a proteinase that is involved in trichomonal cytotoxicity
[45,46]. It has been proposed that the induction of host cell
apoptosis by several trichomonad CPs may also aid the
development of a persistent infection [52].
2.5. Immune evasion mechanisms
Probably due to chronic vaginal infections caused by T.
vaginalis and the constantly changing microenvironment that
it encounters during the menstrual cycle, this parasite has
evolved defenses against the majority of host immune
responses, using a variety of immune evasion mechanisms:
i) To escape from host immune responses T. vaginalis
proteinases degrade human immunoglobulins (IgG, IgM,
and IgA) [53]. A 60 kDa T. vaginalis CP may play a dual
role for parasite survival by degrading immunoglobulins
and supplying nutrients to the parasites by the degradation
of hemoglobin [1]. Additionally, TvCP39 has also been
reported to degrade IgA, IgG, and IgM molecules [45].
ii) In the absence of specific antibodies, the complement in
human menstrual blood kills the parasite. The resistance
to complement in iron-rich conditions contributes to
microbial pathogenesis. A CP up-regulated by iron that
degrades C3b on the trichomonal surface is likely
responsible for this mechanism [1,13].
iii) Two surface immunogens (P270 and P230) have been
reported to be involved in phenotypic variation. For P230,
the phenotypic variation is based on changes in the
accessibility of the epitope to antibody binding, although
the molecule is localized on the parasite surface at all
times. In contrast, the surface expression of P270 is related
to the presence of a dsRNA virus, iron concentration, and
phosphorylation. P270 displays the structural organization
of classic eukaryotic transmembrane proteins [1,21] and
has been found among the surface proteins recently
identified using a proteomic approach [54].
iv) Molecular mimicry also occurs in T. vaginalis by deco-
rating the parasite membrane with molecules homolo-
gous to host proteins, such as several of the parasite
adhesins (AP65, AP51 and AP33) that are homologous to
host metabolic enzymes (malic enzyme and the succinyl-
CoA synthetase a and b subunits, respectively) [13].
v) T. vaginalis can also coat itself with host plasma proteins
to avoid being recognized as foreign by the host immune
system [1].
vi) The continuous secretion of copious amounts of highly
immunogenic soluble proteins into the vagina may
neutralize antibodies and cytotoxic T lymphocytes, short-
circuiting specific anti-T. vaginalis defense mechanisms
[1] and facilitating the persistence of T. vaginalis.
vii) T. vaginalis induces neutrophil recruitment mediated via
the IL-8 pathway in response to activation by live T. vag-
inalis [55]. However, live T. vaginalis also induces
neutrophil apoptosis via ROS-dependent caspase-3
 E.E. Figueroa-Angulo et al. / Microbes and Infection 14 (2012) 1411e1427 1417

activation and the reduction of Mcl-1 expression [56],
contributing to the resolution of inflammation but not the
disease. Moreover, live T. vaginalis also induces macro-
phage apoptosis via the activation of cytochrome c/cas-
pase-3/p38 MAPK pathway, causing anergy of local
immune milieu. However, it is still unknown whether
parasite direct contact or a release factor is involved. The
role of p38 as final executor of macrophage apoptosis by T.
vaginalis is unique. These novel signaling mechanisms
might confer insight into the possible molecular mecha-
nism for the immune evasion of macrophage attack by T.
vaginalis, and as possible chemotherapeutic targets [57].
changes in pH, temperature, Fe2þ, Zn2þ, and polyamine
concentration [5]. An example of the effects of changes in
nutrient composition on the expression of several virulence
factors during the growth of T. vaginalis is the surface expres-
sion of TvCP30 in a colchicine-synchronized culture grown for
24 h in regular culture medium (20 mM iron) (Fig. 2B). The
TvCP30 surface expression is similar to the one observed in
parasites grown in iron-depleted conditions (Fig. 2C).
3.1. The effect of iron and cellular contact in the
expression of virulence genes

Since the vaginal environment is constantly changing under

3. The effects of environmental factors (iron, polyamines,
zinc, and cellular contact) on the expression of T. vaginalis
virulence genes
The T. vaginalis genome contains genes coding for a great
number of protein families, which may explain the adaptation of
this protist parasite to different host environments including
the influence of the menstrual cycle, it is reasonable to
consider that both survival and the establishment of infection
by the pathogen will depend on the ability of T. vaginalis to
adapt to such changes, including variations in host iron levels
and response to cellular contact [7,19,20,58].
T. vaginalis has a fermentative metabolism in which cyto-
plasmic and hydrogenosomal iron-sulfur proteins play

Fig. 2. Surface expression of TvCP30 during growth in a colchicine-synchronized culture (A, B) and under distinct iron concentrations (C). For synchronization,
parasites were treated with 1.5 mM colchicine for 10 h, transferred to fresh medium for 24 h, and then 3H-thymidine was added. Panel A. To track DNA synthesis,
a growth kinetic was performed taking samples every 2 h for 24 h. Panel B. The surface expression of TvCP30 was followed by indirect immunofluorescence with
the anti-TvCP30 antibody (green) and propidium iodide as counterstain (red) [29] using paraformaldehyde-fixed non-permeabilized parasites. In the first 10 h of
parasite growth, four cell duplications, five peaks of incorporation of radioactivity (Panel A), and strong surface fluorescence of TvCP30 in all parasites are
observed (Panel B, pictures from 2 to 10 h), being the highest at 10 h. After that, parasites lost synchrony and the next DNA synthesis occurred at 20 h of growth
(Panel A). Additionally, the surface TvCP30 label was markedly reduced (Panel B, pictures from 14 to 22 h), at similar levels shown by parasites grown in iron-
depleted conditions (panel C, c and d pictures; L) as compared with parasites grown in iron-rich conditions (Panel C, a and b pictures; H) that no TvCP30 surface
label was observed, similar to the label observed at the beginning of the growth kinetic with an asynchronous culture (Panel B, picture from 0 h). These data
suggest that changes in the nutrient composition affects protein surface expression in T. vaginalis.
1418 E.E. Figueroa-Angulo et al. / Microbes and Infection 14 (2012) 1411e1427
a crucial role. T. vaginalis requires up to 300 mM iron for an
optimal metabolism and multiplication in culture [59]. Thus,
T. vaginalis possesses many iron uptake systems like the
presence of a 136 kDa receptor for binding host holo-Lf and
receptors for cytochrome c, hemoglobin, heme, and adhesins
for erythrocytes and epithelial cells. These cells are also the
sources of iron for T. vaginalis [60].
Iron is an essential element for growth and maintenance of T.
vaginalis infection and plays an important role in the
hosteparasite interaction, stimulating cytoadherence and
complement resistance, but reducing cytotoxicity and induction
of host cell apoptosis. Iron modulates the expression of several
virulence genes in T. vaginalis including the adhesins that are up-
regulated and several CPs that are down-regulated by iron
[19,58].
In the T. vaginalis genome, 446 proteases have been
described: 6 asparagine, 17 threonine, 80 serine, 123 metal-
lopeptidases and 220 CPs [5]. Many CPs play important roles
in the virulence of parasites and are involved in cytoadherence
[27e30], cytotoxicity [42,45,46], disruption of the host cell
membrane cytoskeleton [9], degradation of the secretory
leukocyte protease inhibitor [61], hemolysis [49], and human
immunoglobulins [53]. Interestingly, the expression, proteo-
lytic activity, and surface localization of certain trichomonad
CPs are differentially modulated by iron, zinc, and polyamines
in the medium [3,6,60], among other environmental signals.
Morphological and proteomic studies focused on T. vaginalis
grown in the presence or absence of iron showed changes in the
shape and the proteome profile depending on the iron concen-
tration. Approximately 600e640 and 540e570 spots were
detected in gels with protein samples from T. vaginalis grown in
iron-rich and iron-depleted conditions, respectively. The
differentially expressed proteins were identified by mass spec-
trometry (MS). Among the proteins expressed by parasites
cultured in iron-depleted medium, 12 were found up-regulated,
19 down-regulated, 11 had their expression abolished, and three
had their expression induced. Actins, a Rab-GTPase, a 70 kDa
heat-shock protein (HSP70), and several CPs are among those
up-regulated and induced proteins in T. vaginalis collected from
iron-depleted cultures. Expression of multiple actin genes in T.
vaginalis seems to suggest a selective advantage toward the
parasite’s adaptation to different environments. Among the
down-regulated proteins are several CPs and PFO [62]. This
information is consistent with the functional data.
The recently reported T. vaginalis active degradome (i.e.,
the protease-resistant parasite extract) identified 27 protein
spots: 13 were identified as unique proteins, including nine
CPs, seven cathepsin L-like proteins (TvCP1, TvCP2, TvCP3,
TvCP4, TvCP4-like, TvCP12, and TvCPT), and two aspar-
aginyl endopeptidase-like (AE-like) or legumain-like CPs,
(TvLEGU-1 and a legumain-like CP). Several of these
proteins are highly immunogenic and can thus potentially
serve as biomarkers for trichomoniasis; among these proteins,
TvCP39 (TvCPT), TvCP4, TvLEGU-1, and TvCP12 have
been identified and characterized as virulence factors [46,63]
(our unpublished data) and several of them are differentially
modulated by iron, zinc, and polyamines [3,6,45,60].
Likewise, contact between the host cell and this protist
triggers a series of intracellular signals, both in T. vaginalis
and in the host cells [4,8], leading to the expression of other
virulence factors and even to a change in parasite morphology.
On contact with mammalian target cells, T. vaginalis displays
an up-regulation of adhesins and a morphological trans-
formation to an ameboid form, which may be considered to be
another virulence trait [7].
Using a subtraction cDNA library enriched for differen-
tially expressed genes from parasites bound to host cells;
several genes encoding proteins related with colonization,
adherence, change in morphology, and gene transcription and
translation, and others with unknown functions were found.
These included the adhesins AP65 and AP33, a-actinin,
enolase, a putative PDI, a phosphoglucomutase family protein,
and a conserved GTP-binding protein (GTP-BP). Several of
these genes were also differentially regulated by iron. These
data may indicate the possibility of at least two distinct
signaling pathways for up-regulation of expression of these
genes, i.e., iron and contact in T. vaginalis [4,20]. Using
a similar strategy, differentially expressed genes from the
immortalized MS-74 VECs were detected in response to initial
trichomonal infection. Several up-regulated genes encoding
proteins associated with cell structure maintenance and
extracellular matrix components, proinflammatory molecules
and apoptosis were identified in VECs. The up-regulation of
four genes (IL-8, MCP-1, Fn, and COX-2) was confirmed [8].
3.2. The effect of polyamines on the expression of
virulence genes
The polyamines (putrescine, spermidine, and spermine) are
simple aliphatic amines that are essential for life in all
eukaryotes and are present in significant concentrations
(0.1e2.29 mM). Polyamines regulate multiple processes vital
for cellular function and maintenance such as apoptosis, cell
division, differentiation, and cellular growth. These ubiquitous
polycations bind to nucleic acids through electrostatic interac-
tions, affecting transcription and translation. In healthy human
females, the levels of putrescine and diamines in vaginal
secretions are undetectable. However, during trichomoniasis,
vaginal fluids have higher levels of putrescine (>2 mM), sug-
gesting that T. vaginalis produces putrescine during infection
[64] that is exchanged for spermidine from the host.
T. vaginalis lacks the de novo polyamine synthesis
pathway, typical of eukaryotes, and acquires spermidine from
VECs and erythrocytes. Polyamine biosynthesis in this para-
site begins with ornithine synthesis by arginine hydrolase and
putrescine is synthesized by ornithine decarboxylase (ODC).
In T. vaginalis, the inhibition of polyamine synthesis increases
T. vaginalis adherence to VECs [65], but reduces trichomonal
cytotoxicity. This could be due to the requirement of poly-
amines for expression of TvCP65 and TvCP39, two surface-
located CPs involved in cytotoxicity [3,42,45,46]. The
expression of TvCP65 and TvCP39 requires polyamines,
possible at the post-transcriptional level by a mechanism
mediated by polyamines [3], perhaps through RNAeprotein
 E.E. Figueroa-Angulo et al. / Microbes and Infection 14 (2012) 1411e1427 1419

Fig. 3. Effect of inhibition of putrescine biosynthesis on tveif-5a mRNA stability. (A) Levels of tveif-5a mRNA by semi-quantitative RT-PCR analysis using total
RNA from parasites grown in the presence or absence of putrescine and treated with actinomycin D and harvested after 0, 1, 2, 3, 6, 8, 12 and 24 h of treatment for
analysis. PCR products were separated by 1.5% agarose gel electrophoresis and stained with ethidium bromide. Panel N, trichomonads grown in normal medium
under basal conditions; panel D, DAB-treated trichomonads; panel DeP, DAB-treated trichomonads were transferred to medium containing exogenous putrescine;
panel DeN, DAB-treated trichomonads were transferred to normal medium; panel NeP, trichomonads grown in normal medium and transferred to medium with
40 mM exogenous putrescine. The internal control was the 112-bp b-tubulin (b-tub) amplicon (data not shown). (B) The half-life of tveif-5a, assessed by tran-
scription blockage using actinomycin D. Trichomonads grown in normal medium (A). DAB-treated trichomonads (-). DAB-treated trichomonads transferred to

medium with 40 mM exogenous putrescine ( ). DAB-treated trichomonads transferred to normal medium (:). Trichomonads were grown in normal medium and
transferred to medium with 40 mM exogenous putrescine (-). Total cellular RNA was isolated, and the levels of remaining tveif-5a and b-tub mRNA measured by
qRT-PCR are consistent. Values are the means  SE from duplicate samples. *p < 0.00014 when compared trichomonads grown in the presence or absence of
putrescine. This figure is part of Fig. 6 taken from reference [66] with permission from the publisher.

interactions, in which the T. vaginalis eukaryotic translation
initiation factor 5A (TveIF-5A) may be involved.
The eIF-5A is known to be a polyamine-dependent factor
that is characterized by a sequence of 11 conserved amino acids
surrounding the lysine residue at position 50 (Ser-Thr-Ser-Lys-
Thr-Gly-Lys*-His-Gly-His-Ala-Lys) that requires post-
translational modification (hypusination) for maturation. The
amino acid hypusine is not present in other proteins, suggesting
an early origin in the evolution of eukaryotes. T. vaginalis has
two tveif-5a genes, tveif-5a1 and tveif-5a2 that are constitu-
tively expressed [44]. However, the stability of tveif-5a mRNA
depends on the presence of polyamines, showing a half-life
reduction in the absence of polyamines that is recovered by
the addition of exogenous putrescine (Fig. 3) [66].
T. vaginalis ODC is susceptible to analogs of ornithine such
as DFMO (a-dl-Difluoromethylornithine) or DAB (1,4-
diamino-2-butanone), which are effective at reducing intra-
cellular polyamine levels. These analogs caused growth arrest
[65,67] and reduction of the levels of trichomonal cytotoxicity.
Thus, the dependence of T. vaginalis on polyamine scavenging
can be exploited for treatment of this disease.
3.3. The effect of zinc in the expression of virulence
genes
Trichomoniasis appears to be asymptomatic in w70% of
men who are considered to be carriers of T. vaginalis.
Symptomatic patients present urethritis, balanoposthitis,
prostatitis, and other symptoms. The content of Zn2þ in
normal concentrations (4.5e7 mM) in the prostatic fluid has
a trichomonicidal effect that helps to limit or resolve the
infection in the majority of men. However, Zn2þ concentra-
tions similar to those found in men with chronic prostatitis
(<1.6 mM) are not trichomonicidal, and the parasite can be
found in the prostatic gland, causing a non-specific acute
inflammation [2].
Recent studies show that Zn2þ alters trichomonal virulence
by affecting its morphology, proteome, and expression of
several of its virulence factors when the DU145 prostatic cell
line is used as a host cell. T. vaginalis grown in 1.6 mM Zn2þ
displays a reduced growth rate and its proteome exhibits 27
differentially expressed proteins. Two fimbrins and a 50 kDa
metalloproteinase were identified among the over-expressed
proteins in the presence of Zn2þ by MS analysis [6]. This
differential expression of the T. vaginalis proteome in the
presence of zinc may play a key role in its survival in the
adverse environment of the male urinary tract, in comparison
with the vaginal environment.
3.3.1. The cytotoxicity of T. vaginalis in the presence of
Zn2þ
T. vaginalis is able to disrupt DU145 prostatic cell mono-
layers in a contact-dependent manner, as with HeLa cells, but
requires a longer period of time to reach complete monolayer
destruction. T. vaginalis cytotoxicity toward HeLa cell
monolayers is not affected by zinc; however, Zn2þ has
a negative effect on trichomonal cytotoxicity toward DU-145
cells [6]. These could be due to a reduction in the amount,
surface expression, and proteolytic activity of CPs involved in
cytotoxicity (TvCP65 and TvCP39) that also bind to prostatic
cells. The possible mechanism of Zn2þ regulation remains
unknown, and work is in progress to identify it.
1420 E.E. Figueroa-Angulo et al. / Microbes and Infection 14 (2012) 1411e1427
 E.E. Figueroa-Angulo et al. / Microbes and Infection 14 (2012) 1411e1427 1421

Table 1 presents a summary of the virulence factors currently
described in the literature for T. vaginalis. The conditions in
which these molecules are expressed and the type of regulation
involved are indicated. It is evident that the same virulence
factor may be involved in several virulence mechanisms, may
have different localizations in the parasite, and may be regulated
by various stimuli or environmental conditions.
4. Mechanisms of the regulation of T. vaginalis virulence
factors at the level of gene expression
A precise regulation of gene expression is essential for all
cells to adapt quickly to environmental and developmental
changes. This response is highly regulated by multiprotein
complexes that determine which genes should be expressed or
silenced. T. vaginalis possesses several mechanisms to regu-
late the expression of virulence factors, particularly CPs and
adhesins among others. Their regulation can be observed at
four different levels: transcriptional, post-transcriptional,
translational, and post-translational regulation (Fig. 4).
4.1. Transcriptional regulation
T. vaginalis possess one of the largest genomes of any
protist parasite, at w160 Mb encoding for w46,000 proteins
[68]. Comparative transcriptomics in T. vaginalis has gener-
ated almost 100,000 expressed sequence tags (EST) from
parasites cultured under seven defined conditions related to
cell cycle, growth, starvation, and pathogenesis (http://trichdb.
org; http://TvXpress.cgu.edu.tw). Analysis of these data
suggests that gene expression is highly regulated in T. vagi-
nalis by stringent differential transcription rate controls, even
for housekeeping genes (for further details, please consult
these articles [5,23]). An example of differential gene
expression is the modulation of protein kinase gene expression
at the mRNA level. These proteins are involved in signal
transduction, signaling networks, and the response of cells to
environmental changes. Unsynchronized normal culture
library expresses 160 kinases in contrast to the 79 kinases
expressed in low-glucose culture [5]. Another example is the
differential gene expression of the seven genes encoding PFO-
like proteins under different iron concentrations. Only two of
the seven pfo-like genes are expressed in the presence of iron,
pfo a is induced and pfo b is constitutively expressed under the
three iron conditions tested [17].
The knowledge of transcriptional regulation in T. vaginalis is
still very limited, although analysis of the T. vaginalis genome
predicted that w75% of protein-coding genes use a metazoan
initiator-like element (Inr) [5] as a promoter, a highly conserved
sequence surrounding the transcription start site (TSS) with the
consensus sequence TCA þ 1Py(T/A). This Inr is recognized
by transcription factors associated with RNA polymerase II
(RNApol II) and is responsible for the selection of the TSS in T.
vaginalis cells. Many of the genes involved in virulence exhibit
one or two copies of this Inr in which the distal element is
usually the functional one [68,69].
Although the majority of the genes coding for the transcrip-
tion factors associated with the pre-initiation complex for
RNApol II have been found in the T. vaginalis genome [5], a 39-
kDa protein (IBP39) has been identified that specifically
recognizes the T. vaginalis Inr sequence. IBP39 may serve as
a bridge between the Inr sequence and the RNApol II pre-
initiation complex to select the TSS. Genomic studies have
identified at least 100 proteins that contain an Inr-binding
domain and structures similar to IBP39. Although the specific
targets of these proteins are currently unknown, this finding
supports the concept of stringent transcriptional regulation in
this protist [68,69].
The transcriptional response to different iron concentrations
has only been described for the ap65-1 gene, which encodes
the AP65 adhesin. This gene presents an iron responsive
promoter that contains a core promoter sequence, a single Inr
and eight closely spaced regulatory elements including three
MRE elements (MRE-1/MRE-2r and MRE2f) in the proximal
promoter and three T-rich sequences. Additional MREs were
found in the distal promoter region of this gene [68e71].
These sequence elements are recognized by three Myb tran-
scription factors in T. vaginalis, TvMyb1, TvMyb2 and
TvMyb3, which are responsible for the regulation of the
expression of ap65-1. Tai’s group demonstrated that the
TvMyb1 and TvMyb2 proteins show antagonistic actions on
the basal and iron-inducible transcription of ap65-1 through
dual recognition and differential promoter selection toward
both the MRE-1/MRE-2r and MRE-2f sites. The TvMyb3

Fig. 4. Mechanisms of gene expression regulation in Trichomonas vaginalis. (A) Transcriptional regulation. Top panel shows the promoter regions of ap65-1 and
tvcp4 genes. In the first case, the graphic shows the initiator element (Inr), the iron responsive sequence between nucleotides 98 to 90, MRE domains, and Myb
proteins that interact with this region. For tvcp4, only the Inr domain is shown. Bottom panel, agarose gels show the amplicons after RT-PCR assays of RNA from
actinomycin D-treated parasites grown in different iron conditions (L, N, and H) and harvested at different times (0, 1, 3, 6, and 12 h) after transcriptional blockage
as described in Fig. 3. The data show that tvpfo a gene is up-regulated by iron at the transcriptional level, whereas the tvcp4 gene is constitutively expressed at the
transcriptional level. Its regulation is at the post-transcriptional level (see section B). The b tubulin gene is used as a loading control. (B) Iron post-transcriptional
regulation. These diagrams illustrate the potential scenarios for post-transcriptional regulation by iron depending of the position of the stem-loop structure (IRE) in
the mRNA. A gene with the IRE at the 50 region such as tvcp4, in low iron concentrations, the RNA binding protein (RBP) recognizes and binds to the IRE stem-
loop structure located at the 50 -region of the tvcp4 mRNA, blocking the entry of ribosomes and the protein translation. In high iron concentrations, RBP doesn’t
bind to the stem-loop structure, allowing binding of ribosomes and the TvCP4 translation occurs. A gene with the IRE at the 30 -UTR such as tvcp12 in high iron
concentrations, the RBP doesn’t bind to the IRE hairpin and the mRNA is degraded. In low iron concentrations, the RBP binds to the stem-loop structure, the
mRNA is protected and the protein translation takes place. (C) Post-translational regulation. A possible mechanism at the post-translational level may involve
proteineprotein interactions between CPs and endogenous CP inhibitors such as Trichocystatin. In the absence of proteineprotein interaction the CP shows
proteolytic activity as a clear band in the zymogram. However, the interaction of Trichocystatin with the CP active site blocks the CP proteolytic activity and no
band is observed in the zymogram. (D) Post-translational regulation. Posttranslational modifications such as phosphorylations (P), glycosylations (G), or hypu-
sinations (H) also help to regulate protein activity as occur in the maturation process of TveIF5a.
1422 E.E. Figueroa-Angulo et al. / Microbes and Infection 14 (2012) 1411e1427
 E.E. Figueroa-Angulo et al. / Microbes and Infection 14 (2012) 1411e1427 1423

protein recognizes only the MRE-1 element to activate basal
and iron-inducible ap65-1transcription. These studies
demonstrated that TvMyb2 and TvMyb3 may coactivate basal
and iron-inducible ap65-1 transcription against TvMyb1
through conditional and competitive promoter entries. All
Myb proteins have R1R2R3 domains and three conserved
amino acids that are predicted to make direct contact with the
classical MRE sequence (Fig. 4A) [68,69,72e74]. It was
proposed that the large number of proteins with Inr-binding
domains and other 400 Myb-DNA binding domain contain-
ing proteins found in the T. vaginalis genome may mediate the
recognition of a wide variety of DNA targets [68,69,72e74].
Recently, studies have identified the presence of three
motifs found in T. vaginalis genes that lack the typical Inr
sequence (w25%). Motif 1 contains the previously described
core promoter sequence of the Inr element, Motif 3 resembles
the metazoan MRE element and is recognized by the nuclear
protein M3BP, and Motif 5 is reminiscent of the Inr element
[68,69] (Fig. 4A). Interestingly, we found Motif 3 in the
upstream region of a gene that encodes an asparaginyl
endopeptidase-like CP that is positively regulated by iron, but
lacks the typical Inr sequence and the iron responsive
promoter described for ap65-1 (unpublished data).
4.2. Iron post-transcriptional regulation
Post-transcriptional mechanisms for controlling protein
synthesis are generally mediated by specific RNAeprotein
interactions. These interactions are essential to coordinate the
synthesis of several proteins involved in iron uptake, storage,
and release as well as heme synthesis [75]. In mammals, iron
homeostasis is regulated by a post-transcriptional mechanism
mediated by RNAeprotein interactions between iron regula-
tory proteins (IRP-1 and IRP-2, found in the cytosol of all
mammalian tissues examined to date) with hairpin-loop
structures, dubbed iron-responsive elements (IREs), found in
the UTRs of the target mRNA coding for the proteins in iron
homeostasis, the IRE/IRP system [76].
When intracellular iron concentrations are low, IRPs bind to
IREs, and the regulatory outcome depends on the position of
the IRE in the mRNA: if the IRE is located at the 50 -UTR, the
binding of the IRP inhibits the initiation of translation, and the
level of the protein product decreases. If the IRE is located at
the 30 -UTR, the binding of the IRP protects the mRNA against
degradation, and the level of the protein increases. When iron
is in excess, the RNA-binding activity of IRPs is inactivated,
IRP2 is degraded, and IRP1 binds a 4Fee4S cluster to become
an active cytosolic aconitase (c-aconitase) [75,76].
T. vaginalis does not have the Krebs citric acid cycle, and it
lacks aconitase activity [60]. However, two atypical IRE-like
hairpin-loop structures identified and characterized in the
mRNAs of two CPs involved in trichomonal virulence, TvCP4
and TvCP12, are differentially regulated by iron at the post-
transcriptional level. Additionally, T. vaginalis cytoplasmic
proteins bind to mammalian IREs. Together, these data
suggest the existence of a post-transcriptional mechanism of
iron regulation mediated by RNAeprotein interactions, as in
the IRE/IRP system [77], suggesting that trichomonad IRE-
like sequences may be the ancestral forms of the RNA stem-
loop structures of the IRE/IRP system [60] (Fig. 4B). It will
be very interesting to identify the trichomonad RNA-binding
proteins that recognize these RNA hairpin structures and
other genes regulated by this post-transcriptional mechanism
(work is in progress to identify these proteins in T. vaginalis).
4.3. Polyamines post-transcriptional regulation through
the eIF-5A Response Element (ERE)
In T. vaginalis, the TveIF-5A factor may be part of a post-
transcriptional regulatory mechanism mediated by polyamines
similar to the one reported for human cyclooxygenase-2 gene
(cox-2), where a mature hypusinated-eIF-5A protein interacts
with a stem-loop RNA structure, an eIF-5A Response Element
(ERE), located at the 30 -UTR of the cox-2 mRNA, affecting its
stability, downstream RNA processing, and translation into
protein [78]. A similar mechanism of post-transcriptional
regulation mediated by RNAeprotein interactions has been
proposed for the polyamine-mediated expression of the cyto-
toxic TvCP39, because an ERE-like stem-loop structure was
found in the 30 -UTR region of its mRNA [46] (unpublished);
work is in progress to test this hypothesis.
4.4. MicroRNAs (miRNA)
The regulatory role of double-stranded RNA has been widely
reported in eukaryotes, along with the proteins involved in this
process. These microRNAs (miRNA), or small interfering
RNAs (siRNAs), and the machinery of RNA interference can
cause the degradation of mRNA or suppress its translation,
regulating distinct cellular process proliferation, differentiation,
apoptosis and response to stress. While components of the
miRNA machinery are present in the T. vaginalis genome, how it
is utilized is still unknown, and siRNA has not been shown
robustly in this parasite either [5,68,69]. However, Huang et al.
(2012) identified some specific miRNAs in deep-branching
unicellular flagellates, including T. vaginalis. Based on the

Fig. 5. Schematic diagram that summarize the information presented in this review shows the different T. vaginalis virulence mechanisms and the diverse
regulatory pathways that are involved. On the outside of T. vaginalis, the different virulence mechanisms are shown, as well as the factors involved in each of the
process and the environmental conditions that affect the expression of these genes (Zinc, Iron and Polyamines). The cytoadherence mechanism shows the
molecules participating in the interaction between the parasite and host epithelial cell. In the immune response evasion, BspA-like proteins are shown, and the
turnover of these molecules on the surface of the parasite is exemplified. In the inside of T. vaginalis, the mechanisms of gene expression regulation at different
levels are shown. In the nucleus, the transcriptional regulation is shown. In the cytoplasm, post-transcriptional regulation mediated by the IRE/RBP, mechanisms of
post-translational regulation involving the potential interaction of a CP to trichocystatin, and the post-translational modifications that lead to glycosylation in the
Golgi apparatus of the TvCP39 are shown. We also show the autophagy mechanism suggested by Meza-Cervantez et al., 2011 [17] to explain the relocalization of
hydrogenosomal proteins to the parasite membrane to carry out its function as adhesins. HYD: Hydrogenosome, VAC: vacuole.
1424 E.E. Figueroa-Angulo et al. / Microbes and Infection 14 (2012) 1411e1427
structure of miRNA and the genome sequence 14 ancient animal
miRNA families and 13 plant-specific families were identified
[79e81]. These miRNAs may play an important role in the
regulation of several highly repeated gene families in the
genome such as the CPs and the BspA-like surface proteins. It
will be very useful to explore whether T. vaginalis has this
regulatory mechanism at work [79e81].
4.5. Post-translational regulation
4.5.1. Cystatins: endogenous inhibitors of cysteine
proteinases in T. vaginalis that may help to control
trichomonad proteolytic activity
The cystatins are reversible inhibitors of papain-like CPs
and form a superfamily of homologous proteins subdivided
into three families: the stefins, the cystatins, and the kini-
nogens. The main functions of the cystatins are to control
undesirable proteolysis and thereby protect cells [82]. These
molecules have been found in nematodes, flatworms, and
arthropods but not in parasitic protozoa in which a novel class
of proteinaceous inhibitors of CPs (ICP) was identified; cha-
gasin from Trypanosoma cruzi is the best example. Similar
ICPs have been found in Leishmania mexicana, E. histolytica,
and bacterial pathogens. The ICPs also inhibit papain-like CPs
in a manner similar to the cystatins, but these inhibitors have
completely different sequences [82].
Parasite cystatins have the characteristic domains of these
proteins necessary for inhibitory activity: a G in the N-
terminal region, the reactive QxVxG domain in the central
region and the PW loop in the C-terminal region. These
inhibitors perform a wide variety of specific functions such as
the control of cathepsin-L-like activity in Haemonchus con-
tortus by Hc-cystatin, the establishment of infection by
inhibiting host CPs by Bm-ICP-2 (Cystatin type-2) of Brugia
malayi, the regulation of Hb degradation by Sm-cystatin in
Schistosoma mansoni, and down-regulation of the major
histocompatibility complex class II (MHC-II) and CD86 by
Ov-CPI in Onchocerca volvulus [82].
Although CP regulation has been studied at the genomic and
proteomic levels, the intrinsic regulation of trichomonad CPs by
endogenous CP inhibitors is poorly understood. Three genes
encoding for cystatin-like inhibitors (TVAG_127040,
TVAG_272260, and TVAG_034880), here dubbed trichocys-
tatins (TC1eTC3) have been identified in the genome of T.
vaginalis. These genes are localized in different contigs and
have a distinct sequence similarity. Interestingly, T. vaginalis
has cystatin-like inhibitors that are characteristic of more
complex organisms rather than the ICP-like inhibitors of
protozoa. The presence of three genes encoding for cystatins in
T. vaginalis suggests the relevance of maintaining the proper
regulation of CPs in organisms that have large numbers of CPs.
Thus, we propose that proteineprotein interactions between the
CPs and distinct trichocystatins at certain points in time and
under specific environmental conditions could be another level
of virulence regulation, at the post-translational level, in addi-
tion to those described thus far in T. vaginalis (Fig. 4C).
However, it cannot be excluded that this parasite may also
use these trichocystatins for several of the mechanisms already
described for other pathogens to enable successful host para-
sitism [83]. It can be proposed that the trichocystatins may
undergo distinct gene expression regulation by environmental
factors, show distinct cellular compartmentalization, and have
distinct CPs as substrates. This type of interactions (CP/
inhibitor) will then perform the appropriate biological func-
tions, particularly in relation to trichomonal virulence
(Fig. 4C). Work is in progress to identify and characterize the
three trichocystatins, expecting to answer these questions in
the near future.
4.5.2. Post-translational modifications (PTM) in T. vaginalis
PTMs such as glycosylation and phosphorylation play
crucial roles in regulating the diverse proteineprotein inter-
actions involved in essentially every cellular process and
therefore are required in every microorganism for its devel-
opment. In silico analysis of several trichomonad genes has
predicted distinct types of PTM (glycosylation (Oe or Ne),
phosphorylation, hypusination, among others) (Fig. 4D) that
were also proposed for several proteins after analysis of the
proteome reference map of T. vaginalis [84]. Furthermore,
based on these analyses it is proposed that T. vaginalis has the
machinery to perform both the Oe and Ne glycosylation of
proteins [85]. Protein phosphorylation is undoubtedly the most
common and best-studied of PTMs. The T. vaginalis kinome is
one of the largest eukaryotic kinomes known, consisting of
w880 genes encoding distinct eukaryotic protein kinases
(ePKs) and w40 atypical protein kinases (aPKs) [5,23]. These
information, suggest that this parasite may perform protein
phosphorylation reactions.
To date, the presence of PTM has been reported for only
a few trichomonad proteins, e.g., TveIF5a [44,66,86], cyto-
skeletal proteins, tubulin, and several virulence factors, P270,
AP120, and TvCP39 [18,21,46]. The hypusination of TveIF-
5A lysine residue was demonstrated. TveIF-5A also
undergoes phosphorylation in serine and tyrosine residues, and
O-glycosylations (Fig. 4D). These PTM are required for the
maturation of TveIF-5A and the acquisition of its RNA-
binding capability, with the possible involvement of a post-
transcriptional regulatory mechanism mediated by poly-
amines [86]. Our unpublished data indicate that the hypusine
residue is required for the specific TveIF-5A-ERE binding
(work in progress). Further studies are required to determine
whether the post-translational modifications in TveIF-5A
change in response to host microenvironmental conditions.
Several virulence factors of T. vaginalis are also PTM-
targeted proteins, e.g., P270, AP120, and TvCP39 [18,21,46].
P270 was found to be highly phosphorylated in iron-rich para-
sites in which remains in the cytoplasm, whereas the adhesins
are exposed on the trichomonad surface. Iron, therefore, plays
a role in modulating the surface localization of P270 in virus-
harboring parasites [21,45]. Another protein that possesses
PTM is the AP120 adhesin, a surface glycoprotein that is present
only in iron-rich parasites [18]. Moreover, the cytotoxic
TvCP39 is N-glycosylated, which is the first glycosylated CP
 E.E. Figueroa-Angulo et al. / Microbes and Infection 14 (2012) 1411e1427
detected in T. vaginalis [45,46,63]. However, we still do not
know whether glycosylation is necessary for TvCP39 activation
or if glycosylation modulates its proteolytic activity. Interest-
ingly, all these proteins (P270, AP120, and TvCP39) are highly
immunogenic; thus, it is possible that their post-translational
modifications may contribute to this property.
5. Concluding remarks
It is interesting to note that, for a single-cell parasite, the
degree of complexity of its cytopathogenicity and the mech-
anisms involved in the gene-expression regulation of virulence
factors discovered to date (Fig. 5) are similar to those observed
for multicellular organisms. These also show the presence of
unique features in this protist parasite. T. vaginalis has several
virulence mechanisms and specific glycoconjugates and
proteins have been identified and characterized as virulence
factors involved in trichomonal cytopathogenicity by different
groups. Some of these molecules are surface proteins such as
adhesins, CPs, BspAs, GP63, porins, phospholipase A,
receptors for Fn, Coll, Lam, plasminogen, Hb, Lactoferrin, etc.
Moreover, genomics, transcriptomic, and proteomic analyses
suggest that many more surface proteins with putative trans-
membranal domains remain to be discovered as additional
virulence factors.
Recent studies in gene expression regulation also suggest that
several of the genes encoding virulence factors respond to
different environmental signals (iron, zinc, polyamines, cell-
contact, and other conditions), using regulatory mechanisms at
transcriptional, post-transcriptional, translational, and post-
translational levels. These responses contributed to the para-
site adaptation to environmental changes. Understanding the
multifactorial nature of trichomonal pathogenesis and its regu-
lation may help to comprehend how the parasite survives for
a long time, in a constantly changing environment during the
menstrual cycle or in the presence of zinc. This knowledge may
help to develop better prevention measurements, an opportune
diagnosis, new treatments for control of the disease, and
subsequent eradication of this sexually transmitted disease.
Acknowledgments
The author’s research related with the topic of this review
was supported, in part, by the CINVESTAV-IPN and by grants
from CONACYT (58611, 68949, 153093, and 162123 to R.A.;
83808 to M.E.A.S.) and from ICyTDF México (ICyT-299 and
ICyT-219 to R.A.; PIFUTP08-150 to M.E.A.S.). E.E.F.A.,
F.J.R.G., J.P.R. and R.E.C.G. were scholarship recipients from
CONACYT-México. E.E.C.G., F.J.R.G. and J.S.C.C. were also
scholarship recipients from ICYTDF. We are grateful to
Alfredo Padilla Barberi for his help with the art design, Q.F.B
Leticia Ávila-González for her technical assistance, and
Martha Aguilar for her secretarial support. We apologize to
colleagues whose research was not discussed based on space
constraints. We thank all the reviewers for their time and help
to improve our manuscript.
1425
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