Experimental Parasitology 147 (2014) 33–40

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Experimental Parasitology
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y e x p r

Full length article

Trichomonas vaginalis induces cytopathic effect on human lung

alveolar basal carcinoma epithelial cell line A549
Daile Meek C. Salvador-Membreve a,☆, Sonia D. Jacinto a, Windell L. Rivera a,b,*
a Institute of Biology, College of Science, University of the Philippines, Diliman, Quezon City 1101, Philippines
b
Molecular Protozoology Laboratory, Natural Sciences Research Institute, University of the Philippines, Diliman, Quezon City 1101, Philippines

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• First report of the effect of T.

A549 cells with attached trichomonads (%)

100
vaginalis on human lung alveolar
80
basal carcinoma epithelial cell line
60
A549.
40

• Trichomonas vaginalis adheres to

20
A549 cells in parasite-density
0

manner. 4.0 4.5 5.0

Log no. of Trichomonads
5.5

• Trichomonas vaginalis cause

cytotoxicity and induces apoptosis Adherent
in A549 cells. 100 60

• Interaction of T. vaginalis with 80

Cytotoxicity index (%)

Cytotoxicity index (%)

40
A549 cells causes cytopathic effect. 60

40
20

20

0 0
4.0 4.5 5.0 4.0 4.5 5.0
Log no. of parasites Log no. of parasites
incubated to A549

Cytotoxic
80

60
Apoptotic Index (%)

40

20

0
Control Treated Trit

Induce apoptosis

* Corresponding author. Fax: +63-2-9205471.

E-mail address: wlrivera@science.upd.edu.ph (W.L. Rivera).
☆ Present Address: Daile Meek C. Salvador-Membreve, Biology Department, College of Science, Bicol University, Legazpi City 4500, Philippines.

http://dx.doi.org/10.1016/j.exppara.2014.10.003
0014-4894/© 2014 Elsevier Inc. All rights reserved.
34 D.M.C. Salvador-Membreve et al./Experimental Parasitology 147 (2014) 33–40

A R T I C L E I N F O A B S T R A C T

Article history: Trichomonas vaginalis, the causative agent of trichomoniasis is generally known to inhabit the genito-
Received 22 June 2014 urinary tract. However, several case reports with supporting molecular and immunological identifications
Received in revised form 10 September have documented its occurrence in the respiratory tract of neonates and adults. In addition, the reports
2014
have documented that its occurrence is associated with respiratory failures. The medical significance or
Accepted 6 October 2014
Available online 13 October 2014
consequence of this association is unclear. Thus, to establish the possible outcome from the interaction
of T. vaginalis with lung cells, the cytopathic effects of the parasites were evaluated using monolayer cul-
tures of the human lung alveolar basal carcinoma epithelial cell line A549. The possible effect of association
Keywords:
Trichomonas vaginalis of T. vaginalis with A549 epithelial cells was analyzed using phase-contrast, scanning electron micros-
A549 cells copy and fluorescence microscopy. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide),
Cytopathic crystal-violet and TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling) assays
Cytoadherence were conducted for cytotoxicity testing. The results demonstrate that T. vaginalis: (1) adheres to A549
Host–parasite interactions epithelial cells, suggesting a density-dependent parasite-cell association; (2) adherence on A549 is through
Human lung cells flagella, membrane and axostyle; (3) causes cell detachment and cytotoxicity (50–72.4%) to A549 and
this effect is a function of parasite density; and (4) induces apoptosis in A549 about 20% after 6 h of in-
cubation. These observations indicate that T. vaginalis causes cytopathic effects on A549 cell. To date, this
is the first report showing a possible interaction of T. vaginalis with the lung cells using A549 mono-
layer cultures. Further studies are recommended to completely elucidate this association.
© 2014 Elsevier Inc. All rights reserved.

1. Introduction this, it could be hypothesized the possible development of tricho-

It is known that Trichomonas vaginalis, a flagellated protozoon,
causes one of the most common non-viral sexually transmitted in-
fections in humans. The disease trichomoniasis may be asymptomatic
to symptomatic involving inflammation and irritation with a frothy
malodorous discharge, abdominal pain, vaginitis and signs of col-
pitis macularis in women and urethritis in men (Fichorova, 2009;
Petrin et al., 1998; Schwebke and Burgess, 2004). Recently, several
studies have shown the association of T. vaginalis with pregnancy
complications, cervical cancer, infertility, atypical pelvic inflamma-
tory disease, prostate cancer and increased transmission of herpes
simplex virus and HIV (Guenthner et al., 2005; Pereira-Neves and
Benchimol, 2007) .
From the four species of Trichomonas known to occur in humans,
T. vaginalis is identified to inhabit the genitourinary tract of men
and women and is thought to be site-specific. However, few case
studies have documented the presence of T. vaginalis outside the
genitourinary tract. It has been identified in the pharynx and lower
respiratory tract of neonates born to mothers with T. vaginalis in-
fection and adults with a history of urogenital contact (Carter and
Whithaus, 2008; Duboucher et al., 2003; Wang et al., 2006) .
Although there is no record of mortality associated with the oc-
currence of T. vaginalis in the lungs (Carter and Whithaus, 2008),
its presence is associated with respiratory distress. It has been de-
scribed as coinfecting agent in adults with pulmonary trichomoniasis,
chronic bronchitis and acute respiratory distress syndrome
(Duboucher et al., 2003, 2007; Martinez-Giron et al., 2008; Press
et al., 2001). Studies have also shown its association with neo-
nates suffering from respiratory tract diseases. In six case studies
reported in literature, all cases of neonates infected with T. vaginalis
in the respiratory tract showed symptoms of neonatal respiratory
disease (Carter and Whithaus, 2008). These authors implied the im-
portance of considering T. vaginalis in differential diagnosis of
respiratory compromised neonates.
The survival of T. vaginalis in the lungs as being microaero-
philic has been questioned (Duboucher et al., 2008). Instead of having
mitochondria, T. vaginalis has hydrogenosomes which are known
to produce molecular hydrogen. However, recent studies on T.
vaginalis hydrogenosomes have shown to contain proteins (Trx, TrxP
and Rbr) involved in oxygen stress response, converting oxygen and
reactive oxygen superoxide species to water (Schneider et al., 2011).
Also, it was shown that T. vaginalis has a sophisticated cytosolic and
hydrogenosomal antioxidant system that combats oxygen stress uti-
lizing enzymes that reduce oxygen to water (Gould et al., 2013). With
monad infection in the lungs. Also, it has been shown that the
occurrence of trichomonads in the lungs is associated with a hypoxic
condition which promotes parasites’ growth (Duboucher et al., 2007,
2008; Maritz et al., 2014).
This study documented the interaction of T. vaginalis with human
lung alveolar basal carcinoma epithelial cell line A549 and at-
tempted to explore the possibility that this organism may cause the
etiology or progression of respiratory distress. In this report, the ad-
herence, cytotoxicity and induction of apoptosis of T. vaginalis on
A549 cells were investigated.
Several studies have shown the in vitro cytopathic effect of T.
vaginalis on cervical and vaginal epithelial cells (Alderete et al., 1995;
Bastida-Corcuera et al., 2005; Fiori et al., 1997, 1999; Gilbert et al.,
2000; Kucknoor et al., 2005, 2007; Singh et al., 2009) and with
various immune cell types (Chang et al., 2006; Kang et al., 2006)
but not on human lung epithelial cell line. To the authors’ knowl-
edge, this is the first study of the possible interaction of T. vaginalis
with human lung epithelial cell line. Data presented in this study
suggest the pathogenic potential of T. vaginalis on the alveolar
epithelium.
2. Materials and methods
2.1. Growth of Trichomonas vaginalis
The T. vaginalis isolate (NMDR 100) used in this study was ob-
tained from the Molecular Protozoology Laboratory, Natural Sciences
Research Institute, University of the Philippines Diliman, Quezon
City, Philippines. Organisms were grown in trypticase-yeast extract-
maltose (TYM) medium with 10% bovine serum (Clark and Diamond,
2002). The parasites were harvested in log phase by centrifuga-
tion and washed with phosphate buffered saline (PBS) solution thrice
before the experiment.
2.2. Cell cultures
Established cell line of human lung adenocarcinoma epithelial
cells A549 was obtained from the American Type Culture Collec-
tion (ATCC, Manassas, VA, USA) and maintained in the Cell Culture
Laboratory of the Institute of Biology, College of Science, Universi-
ty of the Philippines, Diliman, Quezon City, Philippines. It was
routinely maintained in HAM’s F12K medium with NaHCO3 and
supplemented with 10% fetal bovine serum, 1% penicillin per ml,
 D.M.C. Salvador-Membreve et al./Experimental Parasitology 147 (2014) 33–40 35

Fig. 1. Cytoadherence of Trichomonas vaginalis to A549 cells. Representative phase-contrast microscopic observations of A549 incubated with live T. vaginalis on (B–D). Ap-
proximately 2.0 × 104 A549 cells/ml were incubated with trichomonads in cell:parasite ratio of 1:1 (B), 1:3 (C) and 1:5 (D) for 30 min. For control experiment, parasites
were not added to the cells (A). (Black arrows point to individual parasites adherent to cells, white arrows point to aggregates of trichomonads adherent to cells). Magni-
fication, 100×.

and 100 pg streptomycin per ml. This was incubated under 5% CO2
in humidified air at 37 °C.
2.3. Adherence assay
The cytoadherence of T. vaginalis to A549 was determined using
the adherence assay as described by Alderete and Garza (1985) with
minor modifications. Approximately 2.0 × 104 A549 cells/ml were
seeded into a 24-well plate and incubated until confluent. All cells
were equilibrated in incubation medium containing two parts com-
plete HAM’s F12K and one part Diamond’s medium for 15 min before
the addition of parasites. The cells were then inoculated with tricho-
monads in a 1:1, 1:3, 1:5 cell:parasite ratios. In the control wells,
the same incubation was used except the parasites were omitted.
After 30 min incubation, the wells were washed with PBS thrice to
remove unbound trichomonads. The plate was air dried and indi-
vidual cells were viewed under the inverted microscope. The
percentage of A549 cells with adherent trichomonads was deter-
mined by counting at least 100 cells (Alderete et al., 1988).
2.4. Cytotoxicity assay
For the evaluation of host cell cytotoxicity mediated by T. vaginalis,
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)
and crystal violet assays were employed.
2.4.1. MTT assay
Viability was assayed in triplicates and repeated three times using
MTT assay (Carlmichael et al., 1987) with minor modifications. Prior
to seeding, cells were washed with PBS and trypsinized. Cells were
seeded at 2.0 × 104 A549 cells/ml into 96-well plate and incubated
overnight at 37 °C, 5% CO2. After overnight incubation, A549 cells
were infected with parasites in cell:parasite ratios of 1:1, 1:3 and
1:5 maintained at 5% CO2 37 °C for 18 h. The wells were gently
washed with PBS four times and cell culture media were replaced.
Then, 20 μL of MTT (5 mg/ml PBS) was immediately added to each
well. After 4 h, the metabolic products were resuspended with 100 μL
of acid isopropanol and the intensity of staining was monitored by
microtiter plate spectrophotometer at 595 nm.
2.4.2. Crystal violet assay
The colorimetric assay for measurement of the host cell cyto-
toxicity caused by T. vaginalis has been described previously (Alderete
and Pearlman, 1984). Approximately 2.0 × 104 A549 cells/ml were
seeded into the 96-well plate and incubated overnight under 5% CO2
at 37 °C prior to the assay. After overnight incubation, A549 cells
were infected with parasites in cell:parasite ratios of 1:1, 1:3 and
1:5 maintained under 5% CO2 at 37 °C for 6 and 12 h. The wells were
washed twice with PBS, and the remaining cells were fixed with
2% formaldehyde. After 10 min, the wells were washed with dis-
tilled water and stained with 0.14% crystal violet dissolved in a 5:2
36 D.M.C. Salvador-Membreve et al./Experimental Parasitology 147 (2014) 33–40

Fig. 2. Scanning electron microscopic observations of the adhesion of T. vaginalis to A549. T. vaginalis adhered to A549 cells through its membrane (A–D, asterisks), flagella
(B, D, white arrows) and axostyle (B, open arrow). T denotes trichomonads and C for A549 cell. Scale bars, 10 μm.

(vol:vol) ethanol-formaldehyde solution. The stained cells were
finally solubilized in 1% sodium dodecyl sulfate (SDS) prepared in
50% ethanol. Individual wells were scanned using a microtiter plate
spectrophotometer at 595 nm. For cytotoxicity determinations, all
measurements of experimental (E) samples were indexed to those
of control (C) samples, which showed no loss of cells from the
microtiter plate wells, and were subtracted from 1.0 and multi-
plied by 100; that is cytotoxicity = (1 – E/C) × 100. Experiments were
done in triplicate and repeated three times.
2.5. TUNEL assay
Apoptotic-like changes were observed using In Situ Cell Death
Detection kit, Fluorescein (Roche Diagnostics) following the man-
ufacturer’s protocol. Cells were seeded at 2.0 × 104 A549 cells/ml into
24-well plate and allowed to grow into confluence. Once cells were
approaching 80% confluence, about 1.0 × 106 T. vaginalis cells/ml were
inoculated into it. For positive control, uninfected wells were treated
with Triton X-100. After 6 h incubation, the plates were washed and
incubated with a TUNEL reaction mixture for 1 h at 37 °C at a hu-
midified chamber in the dark. Fluorescence microscopy was
employed to detect fluorescein labels incorporated in the cells. The
apoptotic cells were stained in green by a fluorescein derivative. The
apoptotic index was calculated as percentage of TUNEL-positive cells
using the following formula: apoptotic index = (number of TUNEL-
positive cells/number of total cell nuclei) × 100 (Hockel et al., 1999)
in at least 500 cells. Experiments were done in triplicate and re-
peated two times.
2.6. Scanning electron microscopy
For clear visualization of the adherence of T. vaginalis on A549,
scanning electron microscopy was employed. About 2.0 × 104 A549
cells/ml were allowed to grow to confluence on coverslips which
were placed in a six-well plate. About 1.0 × 105 T. vaginalis cells/ml
were added into the wells. After 6 h interaction, the cells were fixed
in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) for 2 h
at room temperature. After fixation, the cells-trichomonads were
washed thrice with PBS and were post-fixed for 5 min at room tem-
perature in 1% OsO4 in cacodylate buffer, dehydrated in ethanol and
 D.M.C. Salvador-Membreve et al./Experimental Parasitology 147 (2014) 33–40 37

Fig. 3. Density-dependent adherence of T. vaginalis to A549 cells. Percentage of A549
cells in increasing density of trichomonads. Approximately 2.0 × 104 A549 cells/ml
were incubated with trichomonads in cell:parasite ratio of 1:1, 1:3 and 1:5 for 30 min.
The mean percentage of A549 cells with at least one adhering trichomonad was de-
termined by counting at least 100 cells.
Fig. 4. Density-dependent cytotoxicity of T. vaginalis on A549. Cytotoxicity was de-
termined by MTT colorimetric assay as described in Materials and Methods.
Approximately 2.0 × 104 A549 cells/ml were incubated with trichomonads in
cell:parasite ratios of 1:1, 1:3 and 1:5 for 18 h.

adherent trichomonads in cell:parasite ratio of 1:1 (33%) which was

critical-point-dried with liquid CO2. Afterwards, 20 nm thick layer significantly different from cell:parasite ratio of 1:5 (91%) which in-
of gold were deposited on the preparations, and were examined with dicates that adherence is a function of parasite density (Fig. 3).
a JEOL 25 SII scanning electron microscope at 15 kV.
3.2. T. vaginalis is cytotoxic to A549 cells
2.7. Statistical analysis
The cytotoxicity of T. vaginalis on A549 monolayers was deter-
Experiments were done in triplicate. Statistical analyses were per- mined in two quantitative assays. In this study, MTT assay was
formed using Instat version 5 (GraphPad Software, Inc., San Diego, modified since it was observed that T. vaginalis, though without mi-
CA, USA). Nonlinear regression was used when comparing para- tochondria, can reduce the yellow MTT into a bluish-black color
site density and time against cytotoxicity index and number of A549 which might be a potential limitation for the absorbance reading.
cells with adherent trichomonads. To determine whether there were Washing with warm PBS and shaking steps were employed to lessen
significant differences between group means, analysis of variance the number of parasite in the wells. The co-incubation of tricho-
followed by Tukey’s tests were done. A p-value of less than 0.05 was monads with A549 cells for 18 h resulted in damage to cells in a
considered statistically significant. density-dependent manner. As shown in Fig. 4, increasing the
parasite-to-host cell ratios increased the cytotoxic effects. Greater
3. Results number of trichomonads (1:5) causes greater cytotoxicity (72.4%)
than those with less number of parasites (21–42%) at 18 h (p < 0.05).
3.1. T. vaginalis attaches to A549 cells The 18 h incubation time was selected since preliminary study has
shown maximum disruption of the monolayers after 24 h (data not
Two methods were employed to investigate the cytoadherence shown). Also, this would obviate the possible effect of significant
of T. vaginalis to A549 cells. A number of trichomonads adhere to multiplication of trichomonads during the experiments.
monolayer as observed under phase-contrast microscope (Fig 1A–D)
and the scanning electron microscope (Fig. 2A–D). It was ob-
served that while several trichomonads remained free-swimming
and demonstrated hit-and-run behavior, most trichomonads settled
and remained attached to the cell. One to five trichomonads were
found to be attached to one A549 cell. Trichomonads attached first
singly then as aggregates, clustering around A549 cells (Fig. 1D). After
washing, a significant number of the trichomonads remained ad-
herent to the cells. Several A549 cells were detached from the plate
creating cell-free areas, the wells contained only few cells after
washing. However, there was no sign of damage in the control wells;
the control cells continued to be confluent, intact and viable (Fig. 1A).
The attachment to A549 cells was very stable since successive washes
did not detach the cells.
Scanning electron micrographs of the cytoadherence of tricho-
monads to A549 cells showed that trichomonads attached
themselves through their membrane (Fig. 2A-D), flagella (Fig. 2B,
2D) and axostyle (Fig. 2B). The A549 cells became rounded and pre-
sented regression (Fig. 2B-D). Fig. 5. Cytotoxicity exerted by T. vaginalis on A549 cells after crystal-violet stain-
ing and spectrophotometric analysis. Damage to A549 monolayers is seen after
The adherence of T. vaginalis was quantified by counting the incubation with T. vaginalis in different times. Cytotoxicity was determined after stain-
number of adherent trichomonads per 100 A549 cells. In the ex- ing the remaining fixed cells in microtiter plate wells and subtracting the ratio of
periment performed, there was less number of A549 cells with absorbance at 595 nm of the experimental (E) to control (C) samples from 1.
38 D.M.C. Salvador-Membreve et al./Experimental Parasitology 147 (2014) 33–40

Fig. 6. Flourescence (right plane) and brightfield (left plane) microscopic images of non- apoptotic A549 cells (A-B) and A549 undergoing apoptosis (C-D). A549 cells were
treated with T. vaginalis for 6 h and subjected to TUNEL assay. Untreated cells (A, B), A549 cells treated with the parasite (C-D) and treated with Triton X-100 (E-F). Mag-
nification, 100×.

The colorimetric assay using crystal violet was also conducted
to study the cytotoxicity effect of T. vaginalis to A549 cells as de-
scribed by Alderete and Pearlman (1984). After incubating A549 cells
with T. vaginalis, the cells were washed several times, stained with
crystal violet, and absorbance of the stained material was read at
595 nm. It was monitored that increasing the A549-to-trichomonad
cell ratios (1:1, 1:3 and 1:5) enhanced the cytotoxic effect (Fig. 5).
Minimal cytotoxicity of trichomonads incubated at the cell:parasite
ratios of 1:1 (11%) and 1:3 (31.2%) are significantly different (p < 0.05)
from that of the ratio of 1:5 (50%).
Microscopic observations of the monolayer showed loss of A549
cells after extended exposure to trichomonads. Cytotoxic effects were
seen as early as 3 h after incubation with trichomonads, and sig-
nificant destruction of monolayer occurred by 12 h. Time-dependent
cytotoxicity caused by T. vaginalis was observed which was statis-
tically significant at 1:5 cell:parasite ratio (Fig. 5).
3.3. T. vaginalis induces apoptosis to A549 cells
The TUNEL (terminal deoxynucleotidyl transferase dUTP nick end
labeling) was employed to determine whether T. vaginalis can induce
apoptosis as part of its pathogenic mechanism to A549 cells. A549
epithelial cells were allowed to grow to confluence in 24-well plates
and were incubated with fresh incubation media (negative control)
and for the treated, 1.0 × 105 T. vaginalis cells/ml (1:5 cell:parasite
ratio) were added to the well. For positive control, uninfected cells
were treated with Triton X-100 (Fig 6E-F). Triton X-100 is known
to cause DNA damage and apoptosis to cells (Strupp et al., 2000).
After 6 h of incubation, the plates were washed with warm PBS,
stained with a TUNEL reagent, and observed under a fluorescence
microscope. The TUNEL assay showed that T. vaginalis induces apop-
tosis on A549 cells. A549 cells showed TUNEL positive nuclei, which
produced green signal indicating apoptotic cells (Fig 6D, 6F). There
 D.M.C. Salvador-Membreve et al./Experimental Parasitology 147 (2014) 33–40
80
* The detachment and rounding of A549 cells after exposure to T.
60
Apoptotic Index (%)
40
* ithelial layer from the basement membrane. Studies have shown that
20
* metabolites (Pindak et al., 1986) and a heat and acid labile cell-
0
Control
Treated Triton X
Fig. 7. T. vaginalis induces apoptosis on A549 cells. A549 cells were treated with T.
vaginalis for 6 hours and subjected to TUNEL assay. Asterisks (*) indicate data that
are statistically significant, p < 0.05 (Tukey’s HSD test). Data are expressed as percent
of apoptotic cells compared to the untreated cells. The number of apoptotic A549
cells staining positive for TUNEL were counted under fluorescent microscope and
the percentage positive for each factor was calculated on the basis of at least 500
cells scored. Bars, SE.
were about 20% apoptotic cells of A549 infected with T. vaginalis
and 57% apoptotic cells in Triton X-100 (Fig 7).
4. Discussion
T. vaginalis is known to be a resident of the genitourinary tract;
however, it has been reported in the respiratory tract of neonates
and adults. Since its occurrence in the lungs has been associated
with pulmonary distress or it acts as co-infecting agent, the inter-
action between T. vaginalis and the human lung alveolar basal
carcinoma epithelial cell line A549 was examined.
T. vaginalis has a highly specific mechanism of recognizing its
host cells, exhibiting species-specific host–parasite interactions.
Because of this host-specific nature of T. vaginalis, there have been
difficulties in relating observations made with in vitro systems to
in vivo systems. To date, no animal model of human trichomonia-
sis exists. Recognizing the limitations of using continuous cell lines,
the human lung alveolar basal carcinoma epithelial cell line A549
was used as a substitute for human lung epithelial cells in this study.
The A549 cell was used because of the difficulty of obtaining primary
and normal cell lines of human lungs. Moreover, A549 cells are squa-
mous (Standiford et al., 1990), and T. vaginalis is known to attach
to squamous epithelial cells.
It is known that adhesion of trichomonads to the epithelial cells
in the vaginal environment is important for establishment of patho-
genesis of the parasite. Results from this study have shown that
trichomonads were able to adhere to A549 cell in a density-
dependent manner. This adherence is further supported by scanning
electron microscopy which showed attachment through flagella, axo-
style and the membrane-to-membrane contact between the cell and
the parasite. In studies on primary cell lines of human vaginal ep-
ithelial cells, trichomonads attached to cells in variable positions.
They appeared to be attached laterally through flagella and in some
cases, in axostyle-containing projections (Furtado and Benchimol,
1998). It was also shown that the adhesion is stable since subse-
quent washing and the process of critical point drying for scanning
electron microscopy did not detach the trichomonads from the cells.
Adhesion of trichomonads to epithelial cells is a complex process
which involves a morphological change from a pear-drop shape to
flat, amoeboid form (Gilbert et al., 2000; Hirt, 2013). Several tricho-
monad surface proteins are also involved (de Miguel et al., 2010);
the best described is the T. vaginalis lipophosphoglycan (TvLG) which
binds to the host cell receptor galectin-1 (Bastida-Corcuera et al.,
2005; Ryan et al., 2011). Recently, it was shown that T. vaginalis se-
39
cretes vesicles known as exosomes which can increase the parasite’s
adhesion ability to the host cells (Twu et al., 2013).
vaginalis observed in this study have also been observed in several
mammalian cell cultures. Mirhaghani and Warton (1996) showed with
electron microscopic analysis that direct contact between tricho-
monads and amnion membrane epithelial cells resulted in destruction
of the target cells, and eventually led to the desquamation of the ep-
the cause of rounding and detachment of cells might be due to me-
chanical stress (Heath, 1981), release of cysteine proteases and acid
detaching factor (Garber and Lemchuk-Favel, 1990) of T. vaginalis.
The cytotoxicity of T. vaginalis to A549 was also quantified using
MTT colorimetric assay and crystal-violet assay. Data from this study
indicate that T. vaginalis is capable of destroying A549 cells. It was shown
that adherence of T. vaginalis to A549 cells produced parasite density-
dependent cytotoxic effects. Several studies have also shown the
cytotoxic activity of T. vaginalis on human vaginal epithelial cells (Gilbert
et al., 2000; Rasmussen et al., 1986), HeLa cells (Alderete and Pearlman,
1984), ectocervical cell line (Bastida-Corcuera et al., 2005; Lustig et al.,
2013) and prostate cell line (Lustig et al., 2013). Lustig et al. (2013) also
showed that cytotoxicity is directly related to adherence of the isolate.
To further explore the effect of T. vaginalis on A549 cells, the
TUNEL assay was performed to identify cells undergoing apopto-
sis. Data presented here demonstrated that T. vaginalis can cause
apoptosis on A549 cells. It was observed that there were 20%
apoptotic A549 cells after 6 h of co-incubation with T. vaginalis.
Studies have shown that apoptosis is one of the pathogenic mecha-
nisms of protozoan parasites to ensure their survival in the host cells
(Huston et al., 2003; van Dijk et al., 2005). Previous works have also
shown that live parasites and cysteine proteases of T. vaginalis induce
apoptosis on human vaginal epithelial cell cultures (Kummer et al.,
2008; Sommer et al., 2005), macrophages (Chang et al., 2006; Kang
et al., 2006) and human neutrophils (Ahn et al., 2008). In addition,
the study conducted by Song et al. (2010) demonstrated that T.
vaginalis can delay the apoptosis of human neutrophils which sug-
gests that T. vaginalis can regulate apoptosis of host cells.
The pathogenic potential of T. vaginalis on the lung has been sug-
gested by some authors. Rebhun (1964) implicated it as a possible
cause of respiratory disease, citing mouth to vagina contact as a mode
of transmission. Duboucher et al. (2008) suggested its potential
pathogenic effect on the alveolar epithelium since T. vaginalis was
found to be in close contact with alveolar macrophages in the
bronchoalveolar lavage fluid. Other trichomonads known to infect
lungs (T. tenax, T. foetus, T. gallinarum and Pentratrichomonas hominis
or T. hominis) are also implicated in respiratory infections (Maritz
et al., 2014; Martinez-Giron et al., 2008).
5. Conclusion
This study is the first to show that T. vaginalis has the ability to
adhere, elicit cytotoxic effects and induce apoptosis on A549 cells. Taken
together, this study showed that T. vaginalis can cause cytopathic effect
on A549 cells. The results suggest the possibility that T. vaginalis can
elicit respiratory distress. Only few reports have documented the oc-
currence of T. vaginalis in the lungs. However, the results of this study
indicate that the presence of T. vaginalis in the lungs should be given
due attention. Continued research defining further the pathogenicity
of trichomonads on A549 cells or lung cells is noteworthy.
Acknowledgments
This work was supported by research grants from the Philip-
pine Council for Health Research and Development of the
40 D.M.C. Salvador-Membreve et al./Experimental Parasitology 147 (2014) 33–40
Department of Science and Technology (to DMSM) and the Com-
mission on Higher Education (to WLR). We are grateful to Pilarita
T. Rivera for the helpful discussions during the preparation of the
manuscript and Davin Edric V. Adao and Eunice Alexis Chun for the
technical assistance.
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