Open Journal of Urology, 2013, 3, 232-245
http://dx.doi.org/10.4236/oju.2013.36044 Published Online October 2013 (http://www.scirp.org/journal/oju)
MG289 in Mycoplasma genitalium Enhances Microbial
Invasion and Bacterial Persistence
in Benign Human Prostate Cells*
Wasia Rizwani1, Leticia Reyes2, Jeongsoon Kim1, Steve Goodison1, Charles J. Rosser1#
1Cancer Research Institute, MD Anderson Cancer Center Orlando, Orlando, USA
2Department of Infectious Disease & Pathology, College of Veterinary Medicine, University of Florida, Gainesville, USA
Email: email@example.com, LREYES@dental.ufl.edu, Jeongsoon.Kim@orlandohealth.com,
Received August 9, 2013; revised September 5, 2013; accepted September 12, 2013
Copyright © 2013 Wasia Rizwani et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Introduction: Recent studies suggest that infectious organisms may facilitate initiation and metastasis of many human
cancers. One infectious organism of interest is Mycoplasma genitalium (Mg), a prevalent organism in humans known to
cause sexually transmitted infection, as well as urethritis and prostatitis. Previous studies have demonstrated that benign,
non-tumorigenic human prostate cells (BPH-1) chronically exposed to M. genitalium led to the malignant transforma-
tion of these cells as demonstrated in in vitro and in vivo models. Based on work from our laboratory, we felt this ma-
lignant transformation revolved around a specific M. genitalium’s ABC transporter (MG289) with homology to M. hy-
orhinis’ ABC transporter, p37. In this study, differences in M. genitalium’s ability to infect and induce a unique pro-
teome conducive to tumoral growth were studied with engineered M. genitalium in which the p37 protein was silent.
Materials and Methods: Wild-type M. genitalium (strain 431c, designated as M. genitalium WT) and MG289 deficient
M. genitalium mutant (strain 260_3, designated as Mg260_3) were used for this study. We studied the infectivity poten-
tial between M. genitalium WT and Mg260_3 upon exposure to BPH-1 cells. Furthermore, we set out to identify a
unique proteome in BPH-1 cells exposed to M. genitalium WT that could explain its ability to induce malignant trans-
formation of benign cells. Validation of selected proteomic targets was carried out by Western blot analysis. Results:
Both M. genitalium WT and Mg260_3 strains showed somewhat similar growth curve when absorbance at 450nm was
matched at day 0. Colony forming units (CFUs) were similar for both strains at the same absorbance. However, the
ability to infect BPH-1 cells was greatly reduced in Mg260_3 compared to the M. genitalium WT (p < 0.001). This was
evident by infectivity assays and confocal microscopy. Proteomic analysis of BPH-1 cells infected with either M. geni-
talium WT or Mg260_3 for 8 hr, 24 hr and 6 days demonstrated a considerable shift in protein expression over unin-
fected BPH-1 cells at each time point. The preponderance of perturbed proteins regulated protein synthesis and protein
processing, triggering endoplasmic reticulum stress. Conclusions: In summary, we demonstrate that Mg260_3, which is
deficient of the phosphonate ABC transporter substrate-binding protein; MG289 (homologue to M. hyorhinis p37), is
less effective in invading and maintaining an intracellular persistence in benign human prostate cells. In addition, dele-
tion of MG289 resulted in altered BPH-1 responses to M. genitalium infection as evidenced by differential proteome
profiling of BPH-1 infected cells.
Keywords: Cancer; ER Stress Proteins; Infection; Mycoplasma genitalium; Prostate; Proteomics
Mycoplasma genitalium (Mg) is the smallest known
self-replicating microorganism capable of living in para-
sitic association with host cells, thus obtaining most of
their nutrients that they cannot synthesize from the host
for survival. M. genitalium can grow in close relationship
with mammalian cells, often without any apparent pa-
thology or symptoms for extended periods of time . M.
genitalium has been implicated in urogenital infections of
both men and women around the world and it is recog-
nized as an emerging cause of sexually transmitted infec-
*The authors declare that they have no competing interests.
opyright © 2013 SciRes. OJU
W. RIZWANI ET AL. 233
tions in the United States . Specifically, M. genitalium
has been identified as an independent etiologic agent of
acute and persistent male non-gonococcal urethritis
(NGU) and the cause for approximately 20% - 35% of
non-chlamydial NGU cases [3,4]. In addition, because of
the urethra’s intimate location within the prostate gland,
M. genitalium has also been linked with chronic persis-
tent prostatitis [5,6].
Several lines of evidence indicate a strong association
between mycoplasmal infection and various cancers. Ge-
nomic instability, such as polyploidy, trisomy, and trans-
locations, has been reported in eukaryotic cells infected
with mycoplasmas [7-9]. For example, M. fermentans
and M. penetrans, which are found in high frequencies in
patients suffering from AIDS, can induce permanent
karyotypic changes in mouse embryo C3H cells and
murine myeloid 32D cells [9,10]. M. fermentans infec-
tion of human peripheral blood mononuclear cells can
also promote immortalization of these cells in culture
. The p37 gene of M. hyorhinis, also called Cypl (ex-
tracytoplasmic thiamine binding lipoprotein, phosphonate
ABC transporter substrate-binding protein), has been im-
plicated to induce a more aggressive cellular phenotype
(i.e., increase invasive ability) in FS9 mouse sarcoma
cells . It is believed that phosphonate ABC trans-
porter substrate-binding protein from M. hyorhinis disrupts
cellular contact of FS9 cells from fibroblasts, thereby
making them more motile and invasive [12,13]. Blocking
M. hyorhinis phosphonate ABC transporter substrate-
binding protein with antibodies directed against phos-
phonate ABC transporter substrate-binding protein or re-
moving mycoplasma infection reduces the invasive po-
tential evident in mammalian cells exposed to M. hy-
orhinis or M. hyorhinis phosphonate ABC transporter
substrate-binding protein [12,13].
Furthermore, recombinant M. hyorhinis phosphonate
ABC transporter substrate-binding protein was shown to
enhance the invasive potential of the human prostate
cancer cells PC-3 and DU145, as well as the human
melanoma cancer cells C8161 and A375M, while the
enhancement in invasion could be abrogated by a specific
neutralizing antibody directed at M. hyorhinis phosphonate
ABC transporter substrate-binding protein . Moreover,
PC-3 and DU145 prostate carcinoma cells that were
chronically exposed to recombinant M. hyorhinis phos-
phonate ABC transporter substrate-binding protein ex-
hibited nuclear enlargement and increased migratory po-
tential that coincided with significant changes in the ex-
pression of genes involved in cell cycle, signal transduc-
tion and metabolism . Lastly after 19 weeks in cul-
ture, M. hyorhinis and M. genitalium infected human be-
nign prostate cells, BPH-1, demonstrated malignant trans-
formation evident in a panel of in vitro assays and con-
firmed by the formation of xenograft tumors in athymic
mice. Associated with these changes was an increase in
karyotypic entropy, evident by the accumulation of chro-
mosomal aberrations and polysomy . Interestingly,
phosphonate ABC transporter substrate-binding protein
from M. hyorhinis has significant homology with phos-
phonate ABC transporter substrate-binding protein from
M. genitalium (MG289) .
Based on these findings illustrating, the importance of
the mycoplasmal phosphonate ABC transporter substrate-
binding proteins, we hypothesized that inactivation of M.
genitalium phosphonate ABC transporter substrate-binding
protein would have dire consequences in the ability of
mycoplasma to exert its effect on benign human cells. In
the present study, we investigated the pathogenic poten-
tial of M. genitalium phosphonate ABC transporter sub-
strate-binding prostate (MG289) on BPH-1 cells by using
a MG289 deficient mutant of M. genitalium (Mg260_3)
that was originally created by global transposon mutagene-
sis and compared it in a panel of in vitro assays to
wild-type Mg (Mg WT) . In concert with previous
findings , loss of the MG289 protein did not alter in
vitro growth characteristics of Mg260_3 when compared
to M. genitalium WT. However, this mutation caused a
significant reduction in the ability of M. genitalium to
adhere to and invade BPH-1 cells. Quantitative proteome
studies of BPH-1 cells infected with Mg260_3 or M.
genitalium WT also revealed that loss of the M. genital-
ium phosphonate ABC transporter substrate-binding sig-
nificantly altered how BPH-1 cells responded to my-
coplasmal infection. Namely, BPH-1 cells infected with
M. genitalium WT exhibited significant changes in proc-
esses that involve regulation of gene expression and re-
pair, and protein processing and modification. Further-
more, BPH-1 cells infected with M. genitalium WT ex-
hibited a different and more robust endoplasmic reticu-
lum stress response than uninfected cells or cells infected
with Mg260_3 that if chronically present could incite and
sustain the growth of human cells.
2. Materials and Methods
2.1. Cell Culture
Human benign prostate cell line, BPH-1 was obtained
from the American Type Cell Culture (Manassas, VA).
Cells were maintained in RPMI media supplemented
with 10% fetal bovine serum, 100 units/ml penicillin and
100 mg/ml streptomycin. All cells were incubated at
37˚C in a humidified atmosphere of 5% CO2 in air. All
culture media were purchased from Invitrogen (Carlsbad,
CA). BPH-1 cells previously tested negative for a panel
of mycoplasmal organisms by PCR (Mycoplasma Detec-
tion Ste from Takara, Japan) was used for the study.
Cells also tested negative for Mg and Mh by standard
PCR. Primers for Mg: forward primer-5’-GGTCATG
Copyright © 2013 SciRes. OJU
W. RIZWANI ET AL.
GACAAACAAATTCAGCTTCAC-3’; reverse primer-5’-
Mh: forward primer-5’-CGATCCAACTGTTCAAGGT-
AATTTTTA-3’; reverse primer-5’-CACTTTTTCTAAA-
CACTCCAACATCAT-3’ were used. PCR was carried
out using 20 μl reaction volume containing 200 ng of
DNA and 200 nM of primers (Promega PCR mix). The
amplification conditions of PCR consisted of initial de-
naturation at 95˚C for 2 min followed by 40 cycles of
denaturation at 95˚C for 45 sec, annealing at 55˚C for 45
sec and elongation at 72˚C for 45 sec min, followed by 1
cycle at 72˚C for 10 min.
2.2. Mycoplasma Preparation and Culture
Stock solutions of both M. genitalium strains were pre-
pared by growing the microorganisms to early log phase
in SP4 broth, and stored frozen at −80˚C. Both strains of
M. genitalium were grown in SP4 media under standard
2.3. Growth Curve
Cultures for colony forming unit (CFU) determination of
inoculum were serially diluted 10-fold to 10-8 in SP4
broth then 20 ml from each sample and its corresponding
dilutions were plated in SP4 broth and Agar plates. Broth
cultures were incubated at 37˚C in ambient air, while
Agar plates were incubated at 37˚C in 5% CO2. Broth
tubes were checked daily for a color change, and the re-
ciprocal of the last dilution to show growth was deemed
the color-changing unit (CCU). In order to confirm
growth of M. genitalium WT and Mg260_3, CCU, which
provide a relative indication of the amount of microbes
present, were determined. Samples were serially diluted
tenfold to 10-8 in SP4 broth in duplicate. Broth tubes
were checked daily for color change, and the CCU was
recorded as the reciprocal of the last dilution to show
growth at 14 days. Agar cultures were incubated for at
least 14 days before colonies were counted. Log-phase
cultures of M. genitalium WT and Mg260_3 with the
same absorbances (i.e., same number of colonies) at 450
nm were diluted to 20 ml to give zero absorbance at 450
nm compared to SP4 blank on day 0 and growth was
observed over a period of 14 days. Absorbance was read
every 24 hrs and plotted graphically. The same absorb-
ances gave same CFUs for both M. genitalium WT and
Mg260_3 cultures. The experiment was carried out in
duplicate in three separate experiments and p value of
0.05 was considered significant.
2.4. Infectivity Assay
BPH-1 cells (105) were inoculated with log-phase cul-
tures (3 × 104 CFU) of M. genitalium WT or Mg260_3 to
determine whether the mycoplasmal protein MG289 al-
tered its ability to attach to and infect human cells .
To quantify intracellular M. genitalium load, the inocu-
lum was removed following 3 hrs incubation for cellular
attachment and entry and replaced with medium con-
taining gentamicin (200 μg/ml, Sigma) for 2 hrs. Infected
cells were washed thoroughly and fresh RPMI media
added to the wells. The ability for M. genitalium to per-
sist in culture media and not be transported into human
cells was also quantified from culture supernatants in
separate wells. Cell fractions or culture supernatants were
collected at 8 hrs, 24 hrs and 6 days post-infection fol-
lowing removal of the inoculums for quantification of
bacterial loads using both CCU and CFU assays. In every
case, intracellular M. genitalium was quantitated by Real-
Time PCR (RT-PCR) and standard PCR using MG289
and p110 primers. Extracellular M. genitalium was ob-
served and quantitated using CFU assay (p < 0.05; Stu-
dent’s t-test). Each assay was performed in duplicate.
2.5. Real-Time PCR/ PCR
Following infections, DNA was extracted from cells us-
ing DNA isolation kit (Qiagen) as per manufacturer’s
instructions. Quantitative reverse transcriptase (RT)-PCR
was carried out using ABI 7300 Real-Time PCR System
(Life Technologies) in a 20 μl reaction volume contain-
ing 1 μl of the DNA, 1 μM of gene-specific TaqMan
primer and probe mix. The primers used were purchased
from Integrated DNA Technologies. Mg p110: Probe-5’-/
mSp/-3’; Primer 1-5’-TGAACCGGAAAGTGCAACA-
AGTGC-3’ and Primer 2-5’-AAACTTCCCTGCATTG-
CTGTTCCC-3’. Human GAPDH: Probe-/56-FAM/AA-
1-5’-CTCCCACCTTTCTCATCCAAG-3’ and Primer
2-5’-ACATCACCCCTCTACCTCC-3’. Relative fold chan-
ges in DNA levels were calculated after normalization to
GAPDH using the comparative Ct method (27). PCR was
also carried out using 20 μl reaction volumes containing
200 ng of DNA, 1 μM of gene-specific TaqMan primer
and probe mix for semi-quantitative analysis. Standard
DNA PCR was carried out for M. genitalium MG289
using primers described earlier and Mg p110 primers-for-
ward primers-5’-AAGGGCAAAACGCAAAGG GA-3’
and p110-reverse primers-5’-TCCCCCTCCAGAAACA
CTGG-3’ for validation of M. genitalium WT and
BPH-1 cells (106) were plated and allowed to attach and
grow for a day. Next, cells were infected with log-phase
M. genitalium WT or Mg260_3 for 3 hrs (initial infection
time, 3 × 105 bacteria per infection), followed by 2 hrs
Copyright © 2013 SciRes. OJU
W. RIZWANI ET AL. 235
incubation with 200 μg/ml gentamicin in fresh media.
Cells were then washed three times with PBS to remove
unbound or non-invading mycoplasma. Fresh media was
added and the cells were grown for a total of 8 hrs, 24 hrs
or 6 days post initial infection time. Cells were washed
with PBS again and displaced by versene, collected and
centrifugation at 1500 × g for 5 min at 4˚C. Infection of
all BPH-1 cell pellets was confirmed by PCR using
MG289 and p110 primers as already described. Cell pel-
lets were stored at −80˚C until processing for proteome
studies. Protein lysates from cell pellets were prepared
using mammalian proteome extraction kit (Calbiochem,
Billerica, MA) according to the manufacturer’s protocol.
Extracted protein samples were purified and concen-
trated using the Ultrafree®-0.5 centrifugal filter device (5
kDa cut-off from Millipore). Protein supernatants were
dissolved in 1 M Urea/0.5M triethylammonium bicar-
bonate buffer and were assayed using CBX kit (from G-
Biosciences). A total of three separate iTRAQ labelling
reactions per infection were processed according to the
manufacturer’s instructions (AB Sciex) and as described
by Alvarez et al. . Briefly, the protein samples were
reduced/alkylated and then digested with trypsin. Pep-
tides were then labelled with iTRAQ reagents as follows;
Uninfected 8 hrs, Uninfected 24 hrs and Uninfected 6
days with isobaric tag 114; M. genitalium WT 8 hrs, M.
genitalium WT 24 hrs, M. genitalium WT 6 days with
isobaric tag 115; Mg260_3 8 hrs, Mg260_3 24 hrs,
Mg260_3 6 days with isobaric tag 116. For each iTRAQ
experiment, peptides labelled with one of the three labels
(114 - 116) were pooled before separation using strong
cation exchange (SCX). The samples were dried and the
peptides were separated using SCX as described by Al-
varez . Thirteen peptide fractions were collected,
dried, and analyzed once by LC-MS/MS using nanoLC-
ESI-LTQ Orbitrap Velos (Thermo Scientific) as previ-
ously outlined in . The data acquired for the 13 frac-
tions were processed and merged for each biological rep-
The raw protein files were processed for peak detec-
tion using Mascot Distiller v2.4 (Matrix Science Inc.,
Boston, MA, USA). The MS/MS settings include single
peak window selection from m/z 113.5 to m/z 117.5. The
protein peak was identified and quantified with Mascot
Daemon v2.3 (Matrix Science Inc.) using NCBInr (Jan
2012) entries for Bacteria (7,982,362 sequences) and
Mammalia (811,620 sequences). The following settings
were used for the search: trypsin as cleavage enzyme;
two missed cleavages; methylthio modification of cys-
teines, iTRAQ (N terminal), iTRAQ (K) were fixed modi-
fications; and methionine oxidation, deamidation (N, Q),
and iTRAQ (Y) were selected as variable modifications.
The mass error tolerance for precursor ions was set to 15
ppm and 0.08 Da for fragment ions. An automatic decoy
database search was also performed to assess the false
positive rate of protein identification. Automatic isotope
correction was carried out using the values supplied with
the AB Sciex reagents. For reporting quantification re-
sults, the protein ratio type used was the “weighted” geo-
metric mean, normalization method was “summed inten-
sities”, outlier removal was “automatic” (Dixon’s method
up to 25 data points, Rosner’s method above 25 data
points), the peptide threshold was “above homology” (i.e.,
peptide score does not exceed absolute threshold but is
an outlier from the quasi-normal distribution of random
scores), and the minimum number of peptides for protein
identification and quantification was two with an ex-
pected value <0.05. The intensity values of the assigned
peptides were summed and the protein ratio calculated
from the summed values.
2.7. Lysate Preparation and Western Blotting
Corresponding protein lysates from uninfected BPH-1
cells and cells infected with M. genitalium WT and
Mg260_3 were prepared using mammalian proteome ex-
traction kit (Calbiochem) as described earlier and 10 μg
of protein was run on polyacrylamide-SDS gel. The pro-
teins were transferred to a nitrocellulose membrane and
immunoblotted with antibodies corresponding to various
proteins identified from the proteomics. Blots were also
probed for ER stress proteins using the ER stress Anti-
body kit (cat# 9956, Cell Signaling Technology, Danvers,
MA). Mouse monoclonal antibody to β-Actin was pur-
chased from Sigma-Aldrich, St. Louis, MO and used as a
2.8. Immunofluorescence and Confocal
BPH-1 cells were plated onto cover slips (2 × 104 cells
per cover slip) and were infected with log phase M.
genitalium WT or Mg260_3 for 3 hrs followed by 200
μg/ml gentamicin treatment for 2 hrs. Cells were washed
with PBS twice and then fixed with 10% buffered-for-
malin for 15 min. Uninfected cells were used as control.
Immunofluorescence was performed as per the protocol
published previously . The primary M. genitalium
antibody (polyclonal anti-rabbit Mg obtained from Dr.
Mary B. Brown, University of Florida, College of Vet-
erinary Medicine) was used at 1:1000 dilution. Secon-
dary antibody was mouse anti-rabbit Alexa Fluor-488
(Molecular Probes, Carlsbad, CA). Control experiments
(i.e., no primary M. genitalium antibody) demonstrated
no cross-reactivity between mouse anti-rabbit secondary
antibody as evident by no detectable staining by secon-
dary antibody alone (data not shown). DAPI (Vector labs,
Burlingame, CA) was used to stain the nuclei. Cells were
visualized with a DM16000 inverted Zeiss scanning con-
Copyright © 2013 SciRes. OJU
W. RIZWANI ET AL.
focal microscope with a 63X/1.40NA oil immersion ob-
2.9. Data Analysis
M. genitalium’s ability to invade human cells was ana-
lyzed by ANOVA followed by Tukey’s test. A value of p
≤ 0.05 was considered significant. Cluster analysis of
iTRAQ data was performed as previously described 
using JMP Genomics software (SAS, Cary, NC). Briefly,
only protein ratios that were deemed to be significantly
different from uninfected cells as determined by Pro
GroupTM algorithm were used in the analysis. Protein
ratios (M. genitalium infected/uninfected control) from
each time point were normalized prior to clustering and
clustering was performed by the method of Ward.
3.1. Growth of Mycoplasma genitalium—Wild
Type and Mutant Strains
Mycoplasma genitalium MG289 mutant clone (Mg260_3,
M. hyorhinis p37 homologue deleted) was created by
TN4001 transposon insertion in the MG289 gene; the
transposon is located 570 bases into the 1158 bp MG289
gene . Disruption of MG289 gene was checked by
PCR (Figure 1(A)). M. genitalium WT was positive for
both MG289 and p110 whereas Mg260_3 was positive
for only p110 gene confirming that MG289 gene in
Mg260_3 is disrupted. In vitro growth properties of M.
genitalium WT and Mg260_3 were assessed in SP4 growth
media by CCU method and optical density measurements
taken at 450 nm (Figure 1(B)). During stationary phase,
both M. genitalium WT and Mg260_3 attained similar
microbial numbers. However, Mg260_3 did not attain
stationary growth until day 7 of culture whereas M. geni-
talium WT achieved stationary phase by day 6 of culture
(Figure 1(B)). Live bacteria were confirmed by CFU
assay (data not shown).
3.2. Mycoplasma genitalium MG289 Facilitates
Microbial Invasion and Persistence in
Previous studies have shown that M. genitalium can in-
vade and persist in endocervical cells . Therefore, we
evaluated the ability of M. genitalium WT and Mg260_3
to invade and persist in BPH-1 cells at 2 hrs, 8 hrs, 24 hrs,
and 6 days post-inoculation. BPH-1 cell lysates collected
at 2 hrs post-inoculation confirmed that bacteria attached
and invaded BPH-1 cells. In follow-up assays assessing
internalized bacteria, these BPH-1 cells exposed to M.
genitalium WT and Mg260_3 and subsequently treated to
remove extracellular M. genitalium had its DNA isolated
and subjected to semi-quantitative PCR using a PCR
Figure 1. Growth curves of M. genitalium (M. genitalium
WT and Mg260_3). (A) PCR confirming the depletion of
MG289 (homologue to M. hyorhinis p37) in the mutant
strain, Mg260_3; (B) Growth curve shows that M. genital-
ium WT reaches stationary phase earlier than Mg260_3 (p <
0.05). Values represent the mean and standard error of one
experiment performed in triplicate. Each experiment was
conducted three times.
primer/probe set specific for the p110 gene, which is
present in both M. genitalium WT and Mg260_3 (Figure
2(A)). At 40 cycles, M. genitalium WT DNA was de-
tected with equivalent intensity at all post-inoculation
time points. This is in contrast to Mg260_3 in which less
PCR product was detected at 2, 8, and 24 hrs post-in-
oculation. There was no amplified DNA detected in
Mg260_3 infected cell lysates obtained from 6 day old
cultures (Figure 2(A)).
We next quantified internalized M. genitalium bacteria
by RT-PCR in BPH-1 cell RNA lysates obtained from a
separate infectivity assay (Figure 2(B)). As shown in
Figure 2(B), significantly more M. genitalium WT were
retrieved from BPH-1 cells at all post-infection time points
compared to Mg260_3 (p < 0.001). Although there was a
significant decline in the DNA copy number of both M.
genitalium WT and Mg260_3 over the course of the ex-
periment, the decline was more pronounced in Mg260_3
suggesting that deletion of MG289 may impact both mi-
crobial adherence and invasion. Viable bacteria were re-
trieved from both M. genitalium WT and Mg260_3 in-
fected cultures (Figure 2(C)) confirming that bacteria that
invaded BPH-1 cells were viable and that M. genitalium
Copyright © 2013 SciRes. OJU
W. RIZWANI ET AL. 237
Figure 2. MG289 (homologue to M. hyorhinis p37) gene
facilitates adherence and invasion of M. genitalium in
BPH-1 cells. (A) PCR showing presence of M. genitalium
within BPH-1 cells. M. genitalium WT could infect cells with
higher efficiency than Mg260_3; (B) DNA copy number as
determined by RT-PCR. M. genitaliumg DNA was normal-
ized to BPH-1 GAPDH DNA. **Indicates a significant dif-
ference between M. genitalium WT and Mg260_3 (p < 0.05);
(C) Extracellular M. genitalium as determined by culture.
Values represent the mean ± SD log CFU of triplicate. Each
experiment was conducted three times.
may be trafficking through BPH-1 cells in a similar fash-
ion as human endocervical cells .
Infection of BPH-1 cells with M. genitalium WT and
Mg260_3 was further examined by confocal microscopy
(Figure 3). For infection experiments, all M. genitalium
cultures were adjusted so that 105 BPH-1 cells were in-
oculated with 3 × 104 CFU of M. genitalium WT or
Mg260_3 that were harvested during log phase growth.
At 5 hrs post-inoculation, both internalized M. genitalium
WT and Mg260_3 were found within the perinuclear
region of BPH-1 cells by microscopy (Figure 3(A)), which
is consistent with previous studies . However, con-
Figure 3. Intracellular location of M. genitalium within
BPH-1 cells. (A) Representative confocal microscopic im-
ages of uninfected and M. genitalium infected (M. genital-
ium WT and Mg260_3) BPH-1 cells at 7 hrs post-inocula-
tion. Images were captured using 63X/1.40NA oil immer-
sion objective with DM16000 inverted Zeiss scanning con-
focal microscope, magnification −120 μm; (B) Images are 3
dimensional representation of infected cells, magnification
siderably more aggregates of M. genitalium WT were
found within BPH-1 cells than Mg260_3. The staining
pattern for M. genitalium was specific since neither anti-
body controls, nor uninfected BPH-1 cells were positive
for M. genitalium immunofluorescence (Figure 3(A), first
panel). Figure 3(B) is a 3 dimensional representation of
M. genitalium WT and Mg260_3 in BPH-1 cells.
3.3. BPH-1 Cells Infected with Mycoplasma
genitalium Exhibit an Altered Proteome
In order to identify the host cell processes that are per-
turbed by M. genitalium infection, we used quantitative
proteome profiling of uninfected, M. genitalium WT in-
fected and Mg260_3 infected BPH-1 whole cell extracts.
The BPH-1 infection and cell harvest scheme is summa-
rized in Figure 4. Proteins from BPH-1 cell extracts that
were harvested at 8 hr, 24 hr, and 6 days post-inoculation
were identified and quantified using Mascot v2.3. The
Venn diagram (Figure 5(A)) illustrates the number of
proteins that significantly changed in abundance in at
least one of the ratios (M. genitalium WT vs . uninfected
control, Mg260_3 vs. uninfected control or M. genitalium
WT vs. Mg 260_3).
Next, we examined the BPH-1 proteome for proteins
that may be affected during microbial invasion such as
the endosome pathways [25,26], ubiquination and pro-
teasome degradation pathways  and proteins involved
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W. RIZWANI ET AL.
Figure 4. Diagram summarizing the proteome experimental
design. BPH-1 cells were subjected to Mg infection (M. geni-
talium WT and Mg260_3) for an initial period of 3hr, fol-
lowed by removal of the inoculum and gentamicin treat-
ment (200 µg/ml) for 2hr to eliminate any remaining bacte-
ria that were not internalized in BPH-1 cells. Infected
BPH-1 cells were then harvested at 8 hr, 24 hr and 6 days
post-inoculation and analyzed by PCR—to confirm pres-
ence of Mg, iTRAQ analysis—to identify proteome, and
Western blot—to confirm targets from proteome.
in SRP-dependent co-translational protein targeting .
BPH-1 cells infected with M. genitalium WT exhibited a
significant increase in endosome specific proteins and
proteins that regulate endosome formation and traffick-
ing. In contrast, the majority of these proteins were de-
creased in Mg 260_3 infected cells. M. genitalium infec-
tion also perturbed proteins involved in the proteasome
degradation pathway and the SRP-dependent co-transla-
tional protein targeting to membrane pathway, which may
have an impact on endoplasmic reticulum function .
The majority of proteins associated with these processes
were significantly increased in BPH-1 cells infected with
M. genitalium WT, but decreased in cells infected with
Two approaches were used to profile the effect of M.
genitalium WT and Mg260_3 infection on BPH-1 cells.
In the first analysis, protein ratios (M. genitalium WT/
control and Mg260_3/control) that reflected a significant
change (p ≤ 0.05), and were accurately identified in all
infected groups were used in this analysis (Figure 5(B)).
Thus, proteins that did not conform to these criteria were
removed before analysis. At 8 hrs post-inoculation, clus-
ter groups 4 and 9 (demarcated with arrows) displayed
the most dramatic differences. Proteins found within clus-
ter group 4 were markedly reduced in M. genitalium WT
infected cells when compared to cells infected with
Mg260_3. Assigned functions of these proteins include
oxidative reduction, perturbation induction of apoptosis,
angiogenesis/cytokine activity/glycolysis, and negative
regulation of cell proliferation. In contrast proteins in clus-
ter group 9 were markedly increased in M. genitalium
WT infected cells and their assigned functions include
virus infectious cycle, cellular protein metabolic process,
transcriptional regulation, and regulation of apoptosis.
Cluster analysis of cell cultures harvested at 24 hrs
post-inoculation, revealed three clusters that were dramati-
cally different between M. genitalium WT and Mg260_3
infected cells (Figure 5(B)). Proteins in clusters 4 and 6
were markedly increased in M. genitalium WT infected
cells, and their assigned functions include apoptosis, cel-
lular response to reactive oxygen species, biosynthetic
processes, cell differentiation, regulation of microtubule
function, protein import into the nucleus, mRNA proc-
essing, and inhibition of transforming growth factor re-
ceptor signaling pathway. Conversely, proteins in cluster
9 were markedly reduced in Mg WT infected cells, and
these proteins are involved in transcription, translation,
and inhibition of DNA damage response or signal trans-
duction by p53 class mediator.
Cluster analysis of infected cultures harvested on Day
6 identified three markedly different cluster groups be-
tween M. genitalium WT and Mg260_3 infected cells
(Figure 5(B)). BPH-1 proteins in cluster group 6 were
markedly reduced compared to cells infected with
Mg260_3, and these proteins are involved in translation,
regulation of defense response, telomere maintenance,
RNA processing, and steroid hormone mediated signal-
ing. Proteins in clusters groups 9 and 10 were markedly
increased in M. genitalium WT infected cells, and their
assigned functions include regulation of cytokinesis, cell-
cell signaling, vesicle-mediated transport, translation, and
long-chain fatty acid biosynthetic process.
The biological response of BPH-1 cells to M. genital-
ium WT and Mg260_3 was also evaluated by determin-
ing the proportion of biological functions or cellular proc-
esses that were significantly perturbed by M. genitalium
infection (Figure 6). Protein ratios that reflected a signifi-
cant difference between M. genitalium WT and Mg260_3
infected cells (p ≤ 0.05) were grouped according to their
assigned biological process based on the blast2go gene
ontology database (http://www.blast2go.com) . Most
notable changes were observed in proteins over the course
of the assay involved in genetic processes such as regula-
tion of transcription, RNA processing and DNA repair
(20% - 22%). Similarly, another 20% of proteins that
were significantly altered between Mg WT and Mg260_3
infected cells are involved in protein processing such as
post-translational modification, enzyme activation, un-
folded protein response, and protein targeting. Though
the infectivity is suppressed in Mg260_3, absence of
MG289 has produced significant changes in the proteome
of infected BPH-1 cells (Figure 6).
3.4. Endoplasmic Reticulum (ER) Stress
Response Increases with Mycoplasma
It is believed that microbial pathogens may manipulate
the ER stress response as a means of prolonging their
intracellular survival within host cells . Since Mg
infection resulted in significant perturbations in the pro-
teasome pathway, which can affect the ER stress response
Copyright © 2013 SciRes. OJU
W. RIZWANI ET AL.
Copyright © 2013 SciRes. OJU
Figure 5. Proteomic analysis reveals a significant number of proteins changing in expression with M. genitalium WT infec-
tion/uninfected control and Mg260_3 infection/uninfected control. (A) Venn diagram illustrates the number of proteins that
significantly changed in abundance in at least 1 of the ratios between comparisons (M. genitalium WT vs. uninfected control,
Mg260_3 vs. uninfected and M. genitalium WT vs. Mg 260_3); (B) Heat maps were generated with protein ratios (M. genital-
ium WT/control and Mg260_3/control) that reflected a significant change (p ≤ 0.05), and were accurately identified in all in-
fected groups used in this analysis.
or unfolded protein response , we assessed the ER
stress response in uninfected and M. genitalium infected
BPH-1 cells by measuring a panel of ER associated pro-
teins by Western blot analysis. We did not observe any
changes in resident ER proteins such as calnexin, BiP,
protein disulfide isomerase, and endoplasmic oxidore-
ductin-1 (data not shown). However, we did observe
changes in proteins that regulate UPR [reviewed in Mar-
tin Hausmann, International Journal of Inflammation 2010]
such as IRE1 (Inositol-requiring enzyme-1), PERK (pro-
tein kinase RNA-like endoplasmic reticulum kinase), and
CHOP/GADD153 (CCAAT/-enhancer-binding protein
homologous protein) (Figures 7(A) and (B)). At 8hr and
24 hr post-infection, both M. genitalium WT and Mg260_3
infected cells exhibited similar responses. Namely, both
M. genitalium WT and Mg260_3 infection induced an
increase in PERK with no appreciable change in the lev-
els of IRE-1 and CHOP. However, by 6 days post-in-
oculation, M. genitalium WT infected cells had a marked
increase in CHOP, whereas cells infected with Mg260_3
displayed a reduction in all three UPR proteins (Figures
7(A) and (B)).
M. genitalium is an obligate parasite of the human uro-
W. RIZWANI ET AL.
Figure 6. Functional distribution of protein ratios that were
significantly different between M. genitalium WT and
Mg260_3 infected cells at 8hr (A), 24 hr (B), and 6 d (C)
post-infection. Gene ontology designations were obtained
from the Blast2go database.
genital tract  that has been implicated in pelvic in-
flammatory disease , acute and persistent male non-
gonococcal urethritis (NGU) [3,4], and chronic persistent
prostatitis [5,6]. Furthermore, M. genitalium may also
play a role in cancer since our group has previously
demonstrated that chronic exposure of human BPH-1 cells
to M. genitalium resulted in malignant transformation of
these benign cells as evident by in vitro and in vivo as-
says . Despite the various reports indicating an asso-
ciation between Mg infection and human diseases, the
means by which M. genitalium can persist in the host and
thus contribute to the pathobiology of these diseases has
not been determined.
In an attempt to identify the minimal essential genes
for life, Glass et al.  established that the phosphonate
ABC transporter substrate-binding protein, MG289 (homo-
logue to Mh p37), in M. genitalium was not necessary for
the survival of this microbe in cell free culture media.
However, given that M. genitalium is an obligate parasite,
retention of MG289 suggests that this gene serves a
critical function during natural infection. Another inter-
esting feature of MG289 is its similarity to the M. hy-
orhinis p37 , which has been shown to enhance ma-
lignancy in various cells lines [12-15]. Based on these
characteristics, we postulated that MG289 serves an im-
portant role in the virulence of M. genitalium. In this
report we have demonstrated that MG289 enhances mi-
crobial attachment, invasion and intracellular persistence
in BPH-1 cells. Further, by using quantitative proteome
profiling, we have been able to identify M. genitalium
WT induced perturbations in BPH-1 cells that pertain to
DNA repair, protein synthesis and protein processing,
which could impact endoplasmic reticulum function.
Overall, our studies with M. genitalium WT were con-
sistent with previous reports [17,18,24]. Although we
observed a slight delay in the exponential growth phase
of Mg260_3 in SP4 media, this mutant did attain a simi-
lar microbial density as M. genitalium WT at stationary
phase, reaffirming that MG289 is not essential for growth
of mycoplasma in cell free culture media . We also
noted that both M. genitalium WT and Mg260_3 could
invade BPH-1 cells, and also exist outside of these cells.
Furthermore, both internalized M. genitalium WT and
Mg260_3 were in association with the nucleus of BPH-1
cells as well as in the cytoplasm. However, deletion of
MG289 affected the efficiency with which M. genitalium
could invade BPH-1 cells, and this may have also im-
pacted the ability of the mutant to maintain an intracellu-
lar existence. For example, the temporal decline in the
number of internalized Mg260_3 was more pronounced
than with Mg WT, but the numbers of extracellular bac-
teria found within BPH-1 cultures were similar for both
strains at all examined time points. M. genitalium WT
and Mg260_3 also induced different proteome profiles
pertaining to intracellular trafficking. For instance, BPH-1
cells infected with M. genitalium WT exhibited greater
concentrations of proteins involved in endosome recy-
cling and late endosome/lysosome pathways. Since we
examine BPH-1 whole cell lysates, these differences in
Copyright © 2013 SciRes. OJU
W. RIZWANI ET AL.
Copyright © 2013 SciRes. OJU
Figure 7. Western blot analysis reveals changes in protein expression at various time points with M. genitalium WT and
Mg260_3 infections of BPH-1 cells. (A) Western blot analysis of ER stress proteins IRE1α, PERK and CHOP; (B) Western
blot densitometry analysis of the above ER stress proteins IRE1α, PERK and CHOP show differences in expression between
M. genitalium WT infected cells when compared to Mg260_3 infected BPH-1 cells.
protein abundance may be reflecting shifts within differ-
ent cellular compartments (from cytoplasm to endosome),
which are required in rapidly proliferating cells (i.e., can-
cer cells) [35,36].
There have been reports suggesting that M. genitalium
localization to the nucleus may be important in microbial
persistence within the host by evading both host defense
and antibiotic mediated killing [37,38]. Intranuclear ex-
istence within host cells may also play a role in M. geni-
talium induced disease including malignant transforma-
tion . Other bacterial pathogens can cause host cell
DNA damage through their association with the host cell
nucleus [39-41]. As already stated, we identified intra-
cellular M. genitalium in association with BPH-1 nucleus,
and this was most pronounced in Mg WT infected cells.
In addition, our proteome studies in M. genitalium WT
infected cells revealed significant differences in the abun-
dance of proteins that regulate transcription, ubiquination,
apoptosis, cell cycle, and DNA repair. These alterations
may be a reflection of disturbed cell functional pathways
that are associated with carcinogenesis and tumor pro-
gression [42,43]. Although our analysis was restricted to
infections of less than one week in duration, the pertur-
bations that we noted in our proteome analysis may be
early indicators of how M. genitalium induces transfor-
mation of BPH-1 cells . Longer-term experiments are
The preponderance of perturbed proteins that regulate
protein synthesis, protein processing, and their transport
prompted us to consider that M. genitalium infection may
be triggering endoplasmic reticulum stress . There-
fore, we screened uninfected and infected BPH-1 cells
for indicators of a UPR by Western blot. Both M. geni-
talium WT and Mg260_3 induced a similar effect, which
was an increase in UPR . However, as the duration of
infection progressed, the pattern of the UPR response
changed between M. genitalium WT and Mg260_3. This
suggests that the UPR is being triggered by other M.
genitalium factors besides MG289, and that the quality of
the response changes with duration of infection. The res-
toration of IRE1 and PERK levels coupled with an in-
crease in CHOP in M. genitalium WT infected cells was
an intriguing finding. This pattern may be an indication
of a successful adaptive response by M. genitalium to an
intracellular existence [46,47]. Indeed, manipulation of
the host UPR by both viruses and bacteria have been
reported, and they can lead to detrimental effects within
the host [48-50]. For example, herpes simplex virus in-
fection induces phosphorylation of PERK/GADD34 and
downstream signaling involving eIF2α, which in turn fa-
cilitates viral production within host cells . A number
of studies on Human cytomegalovirus (HCMV) indicate
W. RIZWANI ET AL.
that HCMV maintains UPR responses that are beneficial
to the virus while simultaneously inhibiting cellular pro-
cesses that are detrimental to them . For instance,
binding of HCMV Pp28 to BiP/GRP78 maintains the
integrity and function of the HCMV assembly compart-
ment . Infection of myeloid cells with Chlam y dia
trachomatis increases CHOP expression and assists in
the production of pro-inflammatory cytokine, IL-23 .
Hence it is not surprising to find that M. genitalium can
also induce an ER stress response, which may be impor-
tant for its survival and extracellular persistence within
Activation of UPR is also associated with infection-
induced carcinogenesis [52,53]. Increased Hspa5 and Chop
expression is specifically associated with Helicobacter
felis induced gastric dysplasia in C57BL/6 mice .
Enhanced CHOP expression is associated with human
papillomavirus (HR-HPV) infection and abnormal p53
expression in squamous cell carcinoma of uterine cervix
. Therefore, the increased expression of CHOP in M.
genitalium WT infected cells, may also be associated
with malignant transformation of BPH-1 cells during
chronic exposure to M. genitalium .
This is the first study to confirm a pathogenic role for
MG289 (M. hyorhinis p37 homologue in M. genitalium)
in benign human prostate cells. We demonstrate that M.
genitalium mutant, MG289, is capable of infecting BPH-
1cells, though it exhibits a much-suppressed ability to
infect when compared to M. genitalium WT. This feature
can be exploited therapeutically to curb mycoplasma
infection. Both M. genitalium WT and Mg260_3 are ca-
pable of altering the proteome profile of infected BPH-1
cells and the pathways that are altered may relate to on-
cogenic transformation. The differences in pathways re-
lated to DNA repair, endosome trafficking and UPR be-
tween the M. genitalium WT and Mg260_3 can further
be explored in carcinogenesis as well as in the effective
eradication of M. genitalium infections.
In summary, we demonstrate that Mg260_3, which is
deficient in the phosphonate ABC transporter substrate-
binding protein; MG289 (homologue to M. hyorhinis p37),
is less effective in invading and maintaining an intracel-
lular persistence in benign human prostate cells. In addi-
tion, deletion of MG289 resulted in altered BPH-1 re-
sponses to M. genitalium infection as evidenced by dif-
ferential proteome profiling of BPH-1 infected cells.
6. Authors Contributions
All authors have read and approved the final manuscript.
WR—Performed experiments, Acquisition of data,
drafting of manuscript.
LR—Interpretation of proteomics data, drafting of
JK—Performed Western blot assays.
SD—Study concept and design, revision of manuscript.
CJR—Study concept and design, supervision, acquisi-
tion of funding and drafting of manuscript.
We thank Dr. Joel Baseman (University of Texas Health
Science Center, San Antonio, TX) for the generous gift
of Mycoplasma genitalium (M. genitalium WT) and Dr.
John Glass (J. Craig Venter Institute, Rockville, MD)
forM. genitalium p37 mutant, Mg260_3. We acknowl-
edge the funding from Department of Defense Congres-
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