Methionine sulfoxide reductase A (MsrA) deficient Mycoplasma genitalium shows decreased interactions with host cells.
ABSTRACT Mycoplasma genitalium is an important sexually transmitted pathogen that affects both men and women. In genital-mucosal tissues, it initiates colonization of epithelial cells by attaching itself to host cells via several identified bacterial ligands and host cell surface receptors. We have previously shown that a mutant form of M. genitalium lacking methionine sulfoxide reductase A (MsrA), an antioxidant enzyme which converts oxidized methionine (Met(O)) into methionine (Met), shows decreased viability in infected animals. To gain more insights into the mechanisms by which MsrA controls M. genitalium virulence, we compared the wild-type M. genitalium strain (G37) with an msrA mutant (MS5) strain for their ability to interact with target cervical epithelial cell lines (HeLa and C33A) and THP-1 monocytic cells. Infection of epithelial cell lines with both strains revealed that MS5 was less cytotoxic to HeLa and C33A cell lines than the G37 strain. Also, the MS5 strain was more susceptible to phagocytosis by THP-1 cells than wild type strain (G37). Further, MS5 was less able to induce aggregation and differentiation in THP-1 cells than the wild type strain, as determined by carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling of the cells, followed by counting of cells attached to the culture dish using image analysis. Finally, MS5 was observed to induce less proinflammatory cytokine TNF-α by THP-1 cells than wild type G37 strain. These results indicate that MsrA affects the virulence properties of M. genitalium by modulating its interaction with host cells.
Article: Signs and symptoms of urethritis and cervicitis among women with or without Mycoplasma genitalium or Chlamydia trachomatis infection.[show abstract] [hide abstract]
ABSTRACT: To study the prevalence, symptoms, and signs of Mycoplasma genitalium and Chlamydia trachomatis infections in women attending a Swedish STD clinic, accessible for both sexes, and in a group of young women called in the cervical cancer screening programme. A cross sectional study among female STD clinic attendees in Orebro and a study among women called for Papanicolaou smear screening. Attendees were examined for urethritis and cervicitis. First void urine and endocervical samples were tested for M genitalium and C trachomatis. The prevalence of C trachomatis and M genitalium in the STD clinic population was 10% (45/465) and 6% (26/461), respectively. Dual infection was diagnosed in four women. In the cancer screening group of women the corresponding prevalence was 2% (1/59) and 0%, respectively. Among the STD clinic attendees there were no significant differences in symptoms (32% v 23%, RR 1.4, 95% CI 0.6 to 3.4) or signs (71% v 50%, RR 1.4, 95% CI 0.9 to 2.3) between C trachomatis and M genitalium infections. Microscopic signs of cervicitis were significantly more common among M genitalium and C trachomatis infected women than in the cancer screening group of women. 56% (15/27) of male partners of M genitalium infected women were infected with M genitalium compared to 59% of male partners of C trachomatis infected women who were infected with C trachomatis (p = 0.80). M genitalium is a common infection associated with cervicitis and with a high prevalence of infected sexual partners supporting its role as a cause of sexually transmitted infection.Sexually Transmitted Infections 03/2005; 81(1):73-8. · 2.85 Impact Factor
Article: Mycoplasma genitalium as a contributor to the multiple etiologies of cervicitis in women attending sexually transmitted disease clinics.[show abstract] [hide abstract]
ABSTRACT: The purpose of this study was to investigate the prevalence of Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis, and Mycoplasma genitalium, in women attending a sexually transmitted disease (STD) clinic, as well as the frequency of coinfections, and relationship of each organism to cervicitis. In this cross-sectional study of 324 women attending Baltimore City STD Clinics, C. trachomatis, N. gonorrhoeae, T. vaginalis, and M. genitalium were detected using nucleic acid amplification tests. Demographic characteristics and risk factors were ascertained. Overall prevalence of infection with C. trachomatis, N. gonorrhoeae, T. vaginalis, and M. genitalium was found to be 11.1%, 4.6%, 15.3%, and 19.2%, respectively. Prevalence in women with cervicitis was 15.8%, 6%, 18.9%, and 28.6% for C. trachomatis, N. gonorrhoeae, T. vaginalis, and M. genitalium, respectively. Percentages of coinfections were high. C. trachomatis and M. genitalium were significantly associated with cervicitis in univariate analysis, but only M. genitalium was significantly associated with cervicitis (AOR: 2.5) in multiple logistic regression models. Knowledge of the statistical association of M. genitalium with cervicitis in this study increases the need for further confirmation of the etiologic significance of this organism with cervicitis in more diverse populations. The high prevalence merits more study and may have implications for diagnosis and treatment of cervicitis.Sexually transmitted diseases 09/2009; 36(10):598-606. · 2.58 Impact Factor
[show abstract] [hide abstract]
ABSTRACT: Seventy-eight men with a history of chronic urethritis were referred for investigation. Of 52 men diagnosed as having persistent or recurrent non-gonococcal urethritis (NGU) at the time of referral, 11 (21%) were infected with Mycoplasma genitalium and three with Chlamydia trachomatis. Men who were M. genitalium-positive had not previously received less antibiotic, in terms of treatment duration, than those who were M. genitalium-negative, suggesting a possible resistance to the antibiotics given. In the current investigation, of 11 M. genitalium-positive men with persistent or recurrent NGU who were treated for four to six weeks with erythromycin, 500 mg four times daily, nine (82%) responded clinically and microbiologically, but later six relapsed without M. genitalium being detected. The results of observing and investigating a patient for about one year, the only one to have concurrent chlamydial and mycoplasmal infections, is presented, a feature being the intermittent persistence of the mycoplasma.International Journal of STD & AIDS 02/2004; 15(1):21-5. · 1.09 Impact Factor
Methionine Sulfoxide Reductase A (MsrA) Deficient
Mycoplasma genitalium Shows Decreased Interactions
with Host Cells
Kishore Das1,2, Georgina De la Garza1,2, Shivani Maffi1,3, Sankaralingam Saikolappan1,2,
1Regional Academic Health Center, University of Texas Health Science Center at San Antonio, Edinburg, Texas, United States of America, 2Department of Microbiology
and Immunology, University of Texas Health Science Center at San Antonio, Edinburg, Texas, United States of America, 3Department of Molecular Medicine, University of
Texas Health Science Center at San Antonio, Edinburg, Texas, United States of America
Mycoplasma genitalium is an important sexually transmitted pathogen that affects both men and women. In genital-
mucosal tissues, it initiates colonization of epithelial cells by attaching itself to host cells via several identified bacterial
ligands and host cell surface receptors. We have previously shown that a mutant form of M. genitalium lacking methionine
sulfoxide reductase A (MsrA), an antioxidant enzyme which converts oxidized methionine (Met(O)) into methionine (Met),
shows decreased viability in infected animals. To gain more insights into the mechanisms by which MsrA controls M.
genitalium virulence, we compared the wild-type M. genitalium strain (G37) with an msrA mutant (MS5) strain for their ability
to interact with target cervical epithelial cell lines (HeLa and C33A) and THP-1 monocytic cells. Infection of epithelial cell
lines with both strains revealed that MS5 was less cytotoxic to HeLa and C33A cell lines than the G37 strain. Also, the MS5
strain was more susceptible to phagocytosis by THP-1 cells than wild type strain (G37). Further, MS5 was less able to induce
aggregation and differentiation in THP-1 cells than the wild type strain, as determined by carboxyfluorescein diacetate
succinimidyl ester (CFSE) labeling of the cells, followed by counting of cells attached to the culture dish using image
analysis. Finally, MS5 was observed to induce less proinflammatory cytokine TNF-a by THP-1 cells than wild type G37 strain.
These results indicate that MsrA affects the virulence properties of M. genitalium by modulating its interaction with host
Citation: Das K, De la Garza G, Maffi S, Saikolappan S, Dhandayuthapani S (2012) Methionine Sulfoxide Reductase A (MsrA) Deficient Mycoplasma genitalium
Shows Decreased Interactions with Host Cells. PLoS ONE 7(4): e36247. doi:10.1371/journal.pone.0036247
Editor: David M. Ojcius, University of California Merced, United States of America
Received December 12, 2011; Accepted March 29, 2012; Published April 30, 2012
Copyright: ? 2012 Das et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Funding was provided by NIH AI08346 and ERAHC research funds. The funders had no role in study design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
Mycoplasma genitalium is a cell wall-less bacterium and a human
pathogen that causes sexually transmitted diseases such as
urethritis in males and cervicitis in females [1,2,3]. It has been
implicated in female reproductive diseases such as endometritis,
pelvic inflammatory diseases and others [4,5,6]. Increasing
evidences suggest that it may also be a cofactor for HIV
transmission . M. genitalium initiates colonization of epithelial
cells in genital-mucosal tissues by attaching itself to host cells
surface . It primarily uses surface proteins (adhesins) P140
(MgpB) and P32 , encoded by genes MG_191 and MG_318
respectively, for cell adherence. This process is assisted by a group
of proteins called cytadherence accessory proteins that include
several high molecular weight proteins (HMW) [8,10,11]. These
proteins facilitate the translocation and positioning of adhesins on
the surface to form the so called ‘attachment organelle’ which
mediates the attachment process. In addition to attaching to host
cells, M. genitalium has the ability to invade the host cells and persist
there indefinitely [12,13]. Recent in vitro studies have shown that
lipid associated membrane proteins (LAMPs) from M. genitalium
induce proinflammatory responses in monocyte derived macro-
phages which play a role in the clinical manifestations of the
During host-pathogen interactions, mononuclear phagocytic
cells (eg.macrophages) initiate the first line of defense against
invading pathogens. These phagocytic cells have an array of
antimicrobial responses which include generation of reactive
oxygen species (ROS) and reactive nitrogen species (RNS) .
Phagocytes use two different pathways to produce the reactive
species. While phagocyte oxidase (NOX2/gp91phox) produces
superoxide (O2?2) [18,19], inducible nitric oxide synthase (iNOS;
NOS2) produces nitric oxide (NO). The superoxide (O2?2)
undergoes a dismutation reaction or reacts with other compounds
to produce hydrogen peroxide (H2O2) and reactive oxygen
intermediates  such as HO?2, -OOH?2, etc. Likewise, reaction
of NO with other compounds produces reactive nitrogen
intermediates (RNI) such as HNO2, NO2?2. O2?2and NO also
reacts to produce the most potent peroxynitrite, (ONOO?2)
[21,22]. In addition to host derived ROS, some bacterial
pathogens produce ROS as a consequence of aerobic metabolism.
Regardless of the source, both ROIs and RNIs have the ability to
PLoS ONE | www.plosone.org1April 2012 | Volume 7 | Issue 4 | e36247
damage macromolecules such as proteins, lipids, carbohydrates
and nucleic acids. Bacteria use the antioxidants to detoxify ROIs
and RNIs. Conventional antioxidants include enzymes like
catalase-peroxidase (KatG), superoxide dismutase (SOD), alkyl
hydroperoxide reductase (AhpR), organic hydroperoxide reduc-
tase (Ohr) and related enzymes. Interestingly, with the exception
of Ohr, M. genitalium, lacks most of these antioxidant enzymes.
This may be the result of its small genome. In M. genitalium,
however, there are two copies of genes encoding Ohr (MG_427
and MG_454) in M. genitalium. We have recently determined the
role of Ohr encoded by MG_454. This protein appears to defend
against oxidative stress in in vitro experiments .
In addition to conventional antioxidants, methionine sulfoxide
reductases (Msr), which specifically reduce the oxidized methio-
nine (Met-O) to methionine (Met) through a reduction reaction
involving thioredoxin, thioredoxin reductase and NADPH ,
also have roles in the detoxification of ROI. The oxidation of
methionine leads to two different sulfoxides namely Met-S-O and
Met-R-O, which are stereo-isomers. To reduce these sulfoxides,
two enzymes, MsrA and MsrB, exist in most living organisms. In
M. genitalium these enzymes are encoded by MG_408 and MG_448.
These two enzymes are unrelated structurally [25,26,27,28] and
studies have demonstrated that MsrA reduces Met-S-O and MsrB
reduces Met-R-O [24,27,29,30]. The organization of msrA and
msrB varies in different bacterial species and four different types of
organization have been observed. The different organizations
include: a) msrA and msrB genes being located individually in
different regions of the chromosome as separate transcription
units, b) msrA and msrB genes located next to each other as separate
genes but co-transcribed as a single transcription unit, c) msrA and
msrB genes fused together as a single gene to produce a single
protein with two domains, and d) trx, msrA and msrB genes fused
together as single gene to produce a single protein with three
domains. Interestingly, few bacteria have multiple copies of the
genes encoding either msrA or msrB or both and few species
completely lack genes coding for both enzymes . In a subset of
bacteria, Msr is encoded by genes that are present in both plasmid
and chromosomal DNA .
Msr activity has been shown to be important in resistingoxidative
stress in bacteria. However, exceptions have been observed in both
A. actinomycetemcomitans  and Mycobacterium tuberculosis . The
absence of MsrA in most bacteria leads to increased susceptibility to
oxidants in vitro [35,36,37,38,39,40,41,42]. This phenotype could
be reversed by complementation of the mutant strain with
functional msrA. Bacteria deficient in MsrB also have a similar
phenotype for oxidative stress, although there are few exceptions as
with MsrA. In addition, MsrA has also been reported to be an
important virulence factor in pathogenic bacteria because the
absence of this protein affects a range of properties like adherence
[36,43,44,45], motility  biofilm formation [46,47], intracellular
survival  and in vivo survival [36,38,48].
Previously, we have shown that MsrA was a virulence
determinant in M. genitalium, as M. genitalium lacking MsrA was
less able to adhere to sheep erythrocytes and to survive in hamsters
.To gain more insights into the mechanisms by which MsrA
affects virulence in M. genitalium, we compared the wild-type M.
genitalium strain (G37) and msrA mutant (MS5) strain for their
ability to interact with cervical epithelial cell lines (HeLa and
C33A) and THP-1 monocytic cells. In studies related to bacterial
pathogenesis, the routine approach has been to complement the
mutant strains to determine the effect of particular gene products.
Unfortunately, lack of integration and replicating plasmids poses
severe restrictions in complementing M. genitalium mutant strains
with other genes. However, we have used appropriate controls
which include an M. genitalium strain, MGRE, which has a
gentamicin resistant gene in an unrelated locus. We have shown
that an msrA mutant strain has reduced ability to influence the
physiology of target cervical epithelial cell lines (HeLa and C33A)
and THP-1 monocytic cells. We also show that the MS5 strain is
more susceptible to phagocytosis by THP-1 cells than wild type G-
37 strain. Finally, we report here that MsrA is localized primarily
in the cytosolic fraction of M. genitalium.
Materials and Methods
M. genitalium G37, the wild type strain, was grown in 100 ml of
SP-4 medium at 37uC in 150 cm2tissue culture flasks (Corning,
NY) until the color of medium changes to orange. msrA mutant M.
genitalium strain (MS5) and M. genitalium strain MGRE, an
uncharacterized strain that has integration of gentamicin resis-
tance gene in one of the mgpa repetitive regions, were also cultured
in SP-4 medium containing 50 mg/ml gentamicin.
Cell lines and their culture
Human cell lines THP-1 (TIB-202), HeLa (CCL-2), C33A
(HTB-31) and mouse cell line RAW 264.7 (TIB-71) were
purchased from American Type Culture Collection (ATCC,
Manassas, VA). THP-1 cells were cultured in RPMI medium
with 10% FBS (HyClone, Logan, UT). HeLa, C33A and RAW
264.7 cells were cultured in Dulbecco’s modified Eagle’s medium
(DMEM) with 10% FBS. Cultures in both media were grown at
37uC in a humid chamber with 5% CO2.
Preparation of M. genitalium strains for infection
Surface adherent mycoplasmas (G37, MS5 and MGRE) were
washed four times with phosphate-buffered saline (PBS; pH 7.2),
scraped with cell scraper (39 cm handle/3 cm blade; Corning,
NY) and collected by centrifugation (20,0006g, 20 min, 4uC)
using a Sorvall RC 5B centrifuge. The bacterial pellets were
resuspended in PBS and passed first though 18G needles and then
through 23G needles to disperse bacterial clumps. The suspensions
were diluted in PBS to A600=1.0. This was further diluted in
appropriate volume of PBS to infect cell lines with different
multiplicity of infection.
A cytotoxic assay based on sulforhodamine B (SRB) was
adapted from Vichai and Kirtikara . In brief, HeLa and C33A
cells were plated in triplicate on 96 well plates (5,000 cells/well) for
24 h, they were then infected with G37 and MS5 bacteria at
different multiplicity of infection. Cells were incubated at 37uC in
5% CO2 for 12 h, followed by fixing for 1 h with 10% cold
trichloroacetic acid (TCA). Plates were then washed five times in
water, air-dried and stained with 0.057% SRB for 30 min. The
plates were washed again four times with 1% acetic acid, air-dried,
and bound SRB was dissolved in 10 mM unbuffered Tris base
(pH 10.5). The absorbance at A510 was determined using a
SpectraMax M5 microplate reader (Molecular Devices, Sunny-
vale, CA). The percent survival was calculated based on the
absorbance values relative to untreated samples.
Integrity of cell lines after infection with M. genitalium strains
G37, MS5, MGRE and heat killed M. genitalium (HKG37) was
assessed using an Olympus FV1000 confocal laser scanning
microscope and by capturing Differential Interference Contrast
(DIC) images with 206objective (NA 0.75) with 488 nm laser.
Mg Interactions with Host Cells
PLoS ONE | www.plosone.org2 April 2012 | Volume 7 | Issue 4 | e36247
CFSE labeling of THP-1 cells
Carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling
of THP-1 cells was done as described by Clanchy et al. .
Briefly, THP-1 cells were pelleted by centrifugation at 1256g for
6 min and resuspended to106cells/ml in PBS. CFSE (1 mM) was
added and the suspension was incubated at room temperature for
10 min. The reaction was quenched by addition of RPMI medium
containing 5% serum followed by washing with PBS containing
1% FBS. CFSE labeled THP-1 cells were seeded in 4 well 1.5
German cover glass chambers (Nunc, Rochester, NY) at 0.56105
cells per well in RPMI medium and infected with M. genitalium
strains G37, MS5, MGRE and HKG37 (MOI 1:5) for 1 h. The
chambers were washed twice with PBS to remove non-adherent
cells, and images of adherent cells were acquired using Olympus
FV1000 confocal laser scanning microscope with 106 objective
(NA 0.40) and 488 nm laser. The number of labeled cells in each
image were counted using the particle plugin of NIH Image J
software. Average cell numbers from five different optical fields
and from three independent experiments were used for determin-
ing the number of adherent mononuclear cells in each infection.
Phagocytic assays were done using color change assay based on
the ability of surviving bacteria to reduce the chemical MTS to
purple formazan using CellTiter 96 Aqueous One Cell Prolifer-
ation Assay kit (Promega). Briefly, 16105THP-1 cells were seeded
on 96 well plates and differentiated by 100 nM of Phorbol-12-
myristate-13-acetate (PMA) for 48 h in RPMI medium containing
10% serum. The differentiated THP-1 cells were replaced with
100 ml of SP-4 and infected with G37 or MS5 or MGRE (MOI
1:10) and incubated at 37uC in 5% CO2for 1 h. Control wells
contained equal amount of bacteria in 100 ml of SP-4 medium.
After 1 h of incubation, the media from the infected wells were
transferred to wells in a fresh 96 well plate. To remove the
adherent bacteria fully, the wells from the infected plate were
rinsed twice with 50 ml of SP-4 and collected in corresponding
wells in the new plate. To maintain equal volumes, 100 ml of SP-4
medium was added to all the control wells. Next, 40 ml of CellTiter
96 Aqueous reagent was added to all wells, incubated for 1 h at
37uC and absorbance was determined at 490 nm using a
SpectraMax M5 microplate reader (Molecular Devices, Sunny-
vale, CA). Absorbance readings obtained for vehicle control wells
(PBS) were used as blank. The difference in absorbance between
the experimental wells (infected) and control wells were calculated
to determine the amount of bacteria phagocytosed by THP-1 cells.
To confirm the color change assay, phagocytosis of G37 or MS5
or MGRE was visualized by staining the bacteria with Fluorescein
isothiocyanate isomer I (FITC; Sigma-Aldrich, St. Louis, MO)
before infection, as described before . For this experiment,
THP-1 cells (16105) were placed on a 4 well 1.5 German cover
glass chamber (Nunc, Rochester, NY) and differentiated with
PMA (100 nM) for 48 h. Bacterial cells were labeled with FITC by
directly resuspending the pelleted bacteria in a solution containing
FITC (FITC 0.1 mg/ml in 0.1 M NaHCO3, pH 9.0) for 1 h at
25uC . Labeled bacterial cells were washed four times with
PBS and resuspended in PBS. The bacterial cells were used to
infect the differentiated THP-1 cells by incubating for 1 h at 37uC
with 5%CO2. After adding ethidium bromide (50 mg/ml), the
infected cells were washed three times gently with PBS. Images of
the cells were captured using an Olympus FV 1000 confocal
scanning microscope with 488 nm laser and standardized settings
(gain, laser, zoom) across all experimental groups.
Reactive oxygen species (ROS) detection
Generation of Reactive Oxygen Species (ROS) was determined
using the fluorescent probe 29, 79-Dichlorodihydrofluorescein
diacetate (DCF-DA). RAW 264.7 cells (16105) cultured in 4 well
1.5 German cover glass chamber (Nunc, Rochester, NY) for 12 h,
were infected with G37, MS5, MGRE and HKG37 (MOI 1:10)
for 1 h at 37uC and 5% CO2. After infection, cells were washed
twice with 16PBS and ROS generation was detected by adding
1 mM DCF-DA for 30 min at 37uC. Images were captured using a
FV 1000 confocal scanning microscope at 488 nm laser setting as
described above. For reproducibility and comparison, all exper-
imental conditions and microscope settings were kept identical for
all the experiments. Image processing and data analysis were done
with NIH Image-J software. Fluorescence values from captured
images of 10 different fields and from three independent
experiments were calculated and the results expressed as arbitrary
Pro-inflammatory cytokines TNF-a and IL1-b released by
THP-1 and HeLa cells in response to infection by M. genitalium
strains G37, MS5 and heat killed G37 (HKG37) cells were
determined by ELISA. First, THP-1 cells (16105) and HeLa cells (
16104) were plated in 96 well plates and infected with
mycoplasma strains mentioned above (MOI 1:10). Supernatants,
after 1 h and 12 h post infection for THP-1 cells and HeLa cells,
respectively, were collected and centrifuged briefly to settle any
floating cell debris. The supernatants were diluted (1:4) using PBS
and TNF-a and IL-1b concentrations were determined using an
ELISA kit (Bioscience, eBioscience.com) following manufacturer’s
Preparation of membrane and cytosolic fractions
Cell membranes and cytosolic fractions of mycoplasma strains
were prepared by osmotic lysis as described by Razin . Briefly
M. genitalium G37 and MS5 cell pellets were resuspended in 2 ml of
0.25 M NaCl. Osmotic lysis of the cells was done by addition of 4–
6 ml of deionized water preheated to 37uC followed by incubating
for 15 min at room temperature. Membranes were collected by
centrifugation 34,0006g for 30 min using a Sorvall Discovery
M120SE centrifuge (Kendro, Asheville, NC). Membranes were
washed twice in PBS and dissolved in b-buffer (0.15 M NaCl,
0.01 M b-mercaptoethanol, 0.05 M Tris-HCl, pH 7.4 diluted to
1:20 in deionized water). The supernatant (cytosol) was concen-
trated by ultrafiltration using AmiconH Ultra-4 Centrifugal Filter
Units with 3 kDa cut off (Millipore, Milford, MA,) and protein
concentrations of all fractions were determined using Pierce BCA
Protein Assay Kit (Thermo Scientific, Rockford, IL).
SDS-PAGE and Western blots were performed following
standard protocols . Proteins (20 mg/lane) from whole cell
lysate, membrane and cytosol fractions of G37 and MS5 were
resolved using NuPAGE 12% Bis-Tris Gel (1.0 mm610 well;
Invitrogen, Carlsbad, CA). Proteins were transferred to Whatman
Protran nitrocellulose membranes (Whatman, Dassel, Germany),
blocked with 5% skim milk and probed with anti-M. pneumoniae
MsrA rabbit serum  [1:1,000 in Tris-buffered saline containing
0.1% Tween-20 (TBST)] or anti-M. pneumoniae elongation factor G
(EF-G) rabbit antiserum (1:2000 in TBST) . After washing
three times for 5 min each with TBST, membranes were
incubated at room temperature for 1 h with horseradish
peroxidase (HP)-conjugated goat anti-rabbit IgG (Sigma Aldrich,
Mg Interactions with Host Cells
PLoS ONE | www.plosone.org3 April 2012 | Volume 7 | Issue 4 | e36247
St. Louis, MO) at a concentration of 1:10,000 in TBST. The blots
were developed with Pierce ECL Western blotting substrate
(Thermo Scientific, Rockford, IL) and the signals were captured
with BioMax Light Film (KODAK, Rochester, NY).
Immunofluorescence staining of MsrA
To examine the localization of MsrA, immuno-staining protocol
was used as described by Wayne et al. . Briefly mycoplasma
strains (G37 and MS5) cells were pelleted as described above and
were fixed in 2% paraformaldehyde for 30 min, permeabilized
with 0.1% Triton X-100 and 0.1% Tween-20 in PBS for 30 s,
followed by blocking with PBS containing 5% BSA and 5%
normal serum. Cells were suspended using a 23G needle followed
by overnight incubation at 4uC with anti-MsrA rabbit serum. The
bacterial cells were washed three times with PBS followed by
staining with Fluorescein anti-rabbit IgG (Vector, Burlingame,
CA) for 2 h in room temperature. The cells were washed three
additional times with sterile PBS, suspended uniformly using a
23G needle and mounted on a glass slide. Images were captured
with an Olympus FV 1000 confocal scanning microscope using a
606objective (NA 1.42) and 488 nm Argon laser settings with an
electronic zoom of 56. Parallel experiments were done with non-
permeabilized cells as controls.
Paired t-test was performed using graphpad prism software.
msrA mutant strain of M. genitalium is less cytotoxic
Cytotoxicity is a virulence mechanism in few pathogenic
mycoplasmas including Mycoplasma pneumoniae [57,58]. To under-
stand if the deletion of msrA gene has any effect on the cytotoxicity
of M. genitalium, we compared the cytotoxic effects of M. genitalium
wild type (G37) and msrA mutant (MS5) strains of M. genitalium by
infecting HeLa and C33A epithelial cell lines with multiple MOIs
and measuring the SRB signal from the culture. Both cell lines
showed decreased cell survival due to cytotoxicity by M. genitalium
strains, although they differed in their responses (Fig. 1A and 1B).
HeLa cells displayed little cell death even at a high MOI of 1:50
and a significant decline in cell survival was noticed only with
MOI of 1:75 and greater. In contrast, C33A cells showed a decline
in cell survival at lower MOI of 1:10. Increased levels of cell deaths
were observed with increased levels of infections in both cell lines,
although a significant portion of cells were still alive even after
infecting these cell lines with an MOI of 1:100. However,
comparison of cytotoxic effects between G37 and MS5 strains
revealed that the latter had significantly lower cytotoxic effects on
both cell lines.
To confirm the results obtained with the SRB assays, the
cytotoxicity of these strains were visually analyzed with a
microscope. As can be seen in Fig. 2, no cell lysis was observed
with the cells treated with PBS (control) or cells infected with heat
killed G37 strain. In contrast, cells infected with live G37, MGRE
and MS5 strains show significant lysis of the cells. Cell lysis by
MS5 is only moderate as compared to G37 strain, thus reflecting
the results observed with the SRB assay. These data demonstrate
that MsrA is an important component required for the cytotoxic
effect of M. genitalium. To investigate whether the cytotoxic effect of
M. genitalium results in necrotic or apoptotic cell death, HeLa cells
infected with G37strain were stained using the Apoptotic/
Necrotic/Healthy Cells Detection kit (PromoKine, Heidelberg,
Germany). This differentiates apoptotic and necrotic cell death by
green and red fluorescence, respectively. Confocal microscopic
observation of the cells revealed (data not shown) that 99% of the
dead cells stained with red fluorescence, indicating that cell death
due to M. genitalium cytotoxicty was primarily through a necrotic
msrA mutant strain of M. genitalium is more susceptible
Phagocytosis of invading pathogens by host macrophages is an
important event in host-pathogen interactions. A recent study
reported the dynamics of M. genitalium phagocytosis by human
monocyte derived macrophages . To assess if there is a
difference between G37 and MS5 strains in phagocytosis by
macrophages, PMA differentiated THP-1 cells were exposed to
these mycoplasma strains and the uptake was assessed using a
color change assay. The results indicate that after 1 h postinfec-
tion, significantly more MS5 bacteria were phagocytosed than
either G37 or MGRE strains (Fig. 3A). To further confirm this
observation, FITC labeled strains of M. genitalium were allowed to
be phagocytosed by THP-1 cells. Confocal analysis (Fig. 3B) of the
THP-1 cells revealed that fluorescent bacteria were higher in the
phagosomes of THP-1 cells infected with MS5 strain than
phagosomes of THP-1 cells infected with G37 and MGRE strains,
confirming the results from color change assays. These observa-
tions suggest that the absence of MsrA leads to enhanced uptake of
M. genitalium by THP-1 cells.
msrA mutant strains of M. genitalium induces high ROS in
Macrophages generate reactive oxygen species (ROS) to combat
microbial pathogens. Observed higher uptake of MS5 (msrA
mutant) than G37 strain by THP-1 cells led us to hypothesize that
MS5 ingested THP-1 cells might generate more ROS than wild
type G37 ingested THP-1 cells. To test this hypothesis, THP-1
cells were infected with the M. genitalium MS5 strain and other
control strains and the generation of ROS was determined by
using fluorescent ROS detector DCF-DA. Due to high ROS
background of THP-1 cells, it was difficult to determine ROS
generated due to infection. Therefore, a RAW264.7 mouse
macrophage cell line, which was reported to have less indigenous
ROS, was used . Consistent with our hypothesis, RAW264.7
cells infected with MS5 strain and stained with DCF-DA displayed
significantly higher levels of fluorescence, than cells infected with
G37 strain or MGRE strain (Fig. 4A and B), indicating that more
ROS was produced by cells infected with MS5. This reconfirmed
that msrA mutant MS5 strain is more susceptible to phagocytosis
and consequently to oxidative killing by phagocytes. Nonetheless,
the possibility exists that a portion of the ROS detected in MS5
infected cells might include ROS generated by mycoplasmas.
msrA mutant strain of M. genitalium is less efficient in
differentiating THP-1 cells
It has been well established that Mycoplasma fermentens has the
ability to differentiate mononuclear cells . In this study, the
ability of M. genitalium to differentiate mononuclear cells by
infecting CFSE stained undifferentiated THP-1 cells with wild
type M. genitalium strain G37 was investigated. THP-1 cells showed
differentiation and as a result the cells adhered to the bottom of the
flasks and culture slides, similar to that seen with cells
differentiated by PMA. To understand if msrA mutant strain
(MS5) has similar ability to differentiate mononuclear cells, THP-1
cells were infected with this strain and the cells were assessed for
their adherence. As shown in Fig. 5A and B, relatively lower
numbers of THP-1 cells infected with MS5 strain adhered to the
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slide surface as compared to G37 infected cells. It was also
observed that the ability of the MS5 strain in differentiating THP-
1 cells was lower than the level of MGRE strain but higher than
the level of the heat killed G37 bacteria. These results suggest that
the absence of MsrA protein affects some components on M.
genitalium that are required for the induction of differentiation of
msrA mutant strain of M. genitalium induces less TNFa
M. genitalium causes inflammation in genital tissues by inducing
proinflammatory cytokines. Since msrA mutant (MS5) strain
exhibited less virulent properties than the wild type (G37) strain,
we tested for the differences between the two strains in inducing
proinflammatory cytokines IL-1b and TNF-a. To determine this,
both HeLa and THP-1 cell lines were infected with G37 and MS5.
Heat killed G37 was used as a control. ELISA assays of cytokines
released by the cells into the medium revealed that M. genitalium
strains induced only negligible amount of IL-1b and TNF-a in
HeLa cells after 12 h postinfection (data not shown). In contrast,
all strains induced significant amounts of IL-b and TNF-a in
THP-1 cells in an infection time of 1 h (Fig. 6). Although there was
a moderate difference between G37 and MS5 strains in the
induction of IL-1b by THP-1 cells (Fig. 6A), MS5 strain exhibited
significantly lower induction of TNF-a (50%) than wild type strain
(G37) (Fig. 6B). In addition to live G37 and MS5 strains,
significant induction of both cytokines were observed by heat killed
G37 in THP-1 cells, which suggests that certain components of M.
genitalium may be sufficient to induce immune response.
Figure 1. Cytotoxic effect of M. genitalium strains on cervical epithelial cells based on cell survival assay. HeLa (A) and C33A (B)
epithelial cells infected with M. genitalium G37 (wild type) and MS5 (msrA mutant) strains at different multiplicity of infections and cells survived was
determined by SRB (sulforhodamine B) assays. Solid bars in figures A and B represent M. genitalium wild type strain G37. Bars with downward stripes
in figures A and B represent M. genitalium msrA mutant strain MS5. Both strains were tested at various MOI (1:0 21:100). *=p#0.05 percent cell
survival of MS5 strain is higher vs G37 strain. Results represent Mean 6 SD of three independent experiments.
Figure 2. Microscopic observation of cytotoxic effect of M. genitalium strains on cervical epithelial cells. HeLa and C33A epithelial cells
were infected with M. genitalium G37 and MS5 strains and analyzed using differential interference contrast at 488 nm in a confocal laser scanning
microscope with 206objective. PBS indicates uninfected control cells; G37, MGRE, MS5 and HKG37 indicate infection of cells with M. genitalium wild
type G37 strain, control strain MGRE, msrA mutant strain MS5 and heat killed G37 bacteria, respectively.
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MsrA is primarily localized to the cytosol
Previous studies have demonstrated that MsrA is localized both
in the cytosol and membrane fractions of bacteria [33,35]. To
determine the location of MsrA within M. genitalium, we prepared
cytosol and membrane fractions from G37 and MS5 strains and
probed these fractions with anti-MsrA antiserum (Fig. 7A). This
antiserum reacted with MsrA and two cross reactive proteins
(CRPs) in the wild type strain and only CRPs in the msrA mutant
strain. The CRPs were found to be due to the reaction of second
antibody with two M. genitalium proteins; hence they are not shown
here. The results (Fig. 7A-I), however, revealed that both
membrane and cytosolic fractions had MsrA but the amount of
MsrA in the cytosolic fraction was several fold higher than the
membrane fraction. To test whether this small amount of MsrA
with membrane fraction was due to its association with membrane,
we probed the same blot with anti-elongation factor G (EF-G;
MP604) antiserum of M. pneumoniae, a marker for cytosolic protein
[55,62] that is 95% identical to EF-G (MG_089) of M. genitalium.
Results (Fig. 7A-II) revealed moderately intense, less intense and
intense bands for the whole, membrane and cytosolic fractions,
respectively, in both strains. Repeated analysis resulted in similar
pattern of reactivity for MsrA and EF-G in both cytosolic and the
membrane fractions. There appeared to be no problem with the
method used for the preparations of membrane and cytosolic
proteins, since SDS-PAGE (Fig. S1) separated proteins showed
distinct patterns for cytosolic and membrane proteins. It is not
clear whether a portion of both proteins are associated with the
membrane of this species or these proteins associate themselves to
the membrane fractions during the preparation of membranes.
However, since MsrA predominates in the cytosolic fraction like
the cytosolic marker EF-G, we presume that MsrA is primarily a
cytosolic protein in M. genitalium.
In addition to biochemical analysis, immunohistochemistry was
also performed to confirm the localization of MsrA by using anti-
MsrA antiserum and fluorescein conjugated second antibody
(Fig. 7B). In this experiment, a detergent was used to permeabilize
M. genitalium G37 and MS5 bacteria. Confocal analysis revealed
that detergent treated G37 strain had significantly enhanced
fluorescence as compared to untreated bacteria. In contrast, very
limited fluorescence in the detergent bacteria and no fluorescence
in the untreated bacteria, respectively, were seen in the msrA
mutant MS5 strain. These results reinforce the observation that
MsrA is primarily a cytosolic protein and that antibodies cross
react with other proteins of M. genitalium as observed in Western
In this study we have investigated the effects of MsrA protein on
M. genitalium’s interactions with host cells using msrA mutant and
wild type strains. We observed that a loss of MsrA affects the
ability of M. genitalium to cause cytotoxicity in host cells.
Cytotoxicity is an important virulence mechanism of some
pathogenic mycoplasmas that infect human and animal hosts,
and one of the major components that induce cytotoxicity appears
Figure 3. Phagocytosis of M. genitalium strains by THP-1 cells. A. Determination of phagocytosis by color change method.
Phagocytosis of M. genitalium strains by THP-1 cells were determined by a change in color after adding MTS solution (Promega) as described under
Materials and Methods section. The solid bars indicate absorbance (A490) of the control wells (Mycoplasmas without THP-1 cells) and striped bars
represent absorbance (A490) of the experimental wells (THP-1 cells infected with mycoplasmas). Results represent Mean 6 SD from three independent
experiments. G37, MGRE and MS5 indicate infection of cells with M. genitalium wild type G37 strain, control strain MGRE and msrA mutant strain MS5,
respectively. *=p#0.05 vs wild type G37. B. Visualization of phagocytosed M. genitalium G37, MGRE and MS5 strains. G37, MGRE and MS5
bacteria were labeled with FITC as described in Material and Methods. FITC and DIC represent fluorescence and differential interference contrast of
the same field. Merge represents overlay of FITC and DIC. PBS indicate uninfected control cells; G37, MGRE and MS5 indicate infection of cells with M.
genitalium wild type G37 strain, control strain MGRE and msrA mutant strain MS5, respectively.
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to be the H2O2released by these species [57,58]. Interestingly,
generation of H2O2 in these species is linked to oxidation of
glycerol by glycerol 3-phosphate oxidase . This is evident from
the disruption of the glpD gene in M. pneumoniae which codes for
glycerol 3-phosphate oxidase . It has been shown that a glpD
mutant of M. pneumoniae is less able to produce H2O2and as a
consequence is less efficient in producing cytotoxicity , thus
indicating a direct relationship between production of H2O2and
cytotoxicity. In addition to glycerol 3-phosphate oxidase, the ABC
transport system is also implicated in the production of H2O2. This
system is critical for transport of glycerol and strains of M. mycoides
having deletion in ABC locus were found to have reduced
cytotoxicity . Extensive studies in this species reveal that
release of H2O2alone is not sufficient enough to cause cytotoxicity
but the released H2O2 by mycoplasma also needs to be
translocated to the host cells . This indicates that a close
contact between mycoplasma and host cells is required for
cytotoxicity in which adherence plays a major role. In this
context, we have previously reported  that msrA mutant M.
genitalium adheres poorly with red blood cells and the reason for the
reduced cytotoxicity by this strain may be the poor adherence.
Alternatively, the possibility exists that absence of MsrA affects
other sites related to production or translocation of H2O2in the
msrA mutant M. genitalium, thus leading to reduced cytotoxicty. It
may be noted that GlpD and ABC transporter proteins are highly
conserved in M. genitalium .
Although M. genitalium is able to survive intracellularly within
cervical epithelial cells [12,13,68], its ability to survive within
phagocytes is limited. A recent study demonstrated that human
monocyte derived macrophages (MDM) phagocytose M. genitalium
within five minutes of post infection and digest all ingested bacteria
within six hours post infection , suggesting that M. genitalium is
susceptible to phagocytosis mediated killing. Thus, MsrA of M.
genitalium is less likely to play a significant role in defending ROS
generated by macrophages. Nevertheless, the observation that the
absence of MsrA in M. genitalium significantly increases the
phagocytosis by THP-1 cells, as compared to wild type control,
is very striking. Detection of significantly higher levels of ROS in
msrA mutant ingested macrophages than wild type ingested
macrophages provides additional support for the increased level
of phagocyotsis by this strain. Since phagocytosis is a process based
upon ligand–receptor interactions, the altered phagocytosis may
indicate that there is a level of alteration of surface molecules that
mediates binding to phagocytic cells in the msrA mutant strain.
This is a possibility, because alterations in surface ligands/
receptors of pathogens that lack MsrA have already been reported
[43,44]. Whereas S. pneumoniae lacking MsrA had a significant
alteration in the ligand that binds GalNAcb1-4Gal receptors of
eukaryotic cells, enteropathogenic E. coli lacking MsrA had
alterations in the ligand that binds with the mannose receptor
. Conversely, the receptors that bind with ECM molecules,
like collagen, laminin and fibronectin, have also been found to be
affected in Streptococcus gordonii that lack MsrA .
The observation that M. genitalium differentiates THP-1 cells
phenotypically more or less similar to the action of PMA (phorbol
12-myristate 13 acetate) on these cells is interesting. To our
knowledge, this is the first such report with M. genitalium. Thus far,
M. fermentens is the only other mycoplasma that has been reported
to differentiate THP-1 cells . It is very likely that this ability
can bestow additional advantages to M. genitalium while infecting
the host, particularly in the modulation of host immune response
which includes generation of ROS and NO. Thus, this property of
M. genitalium may constitute an important virulence mechanism
and identification of M. genitalium molecules that induce differen-
Figure 4. Generation of Reactive Oxygen Species (ROS) by
phagocytic RAW264.7 cells upon infection with M. genitalium
strains. A. Confocal images of RAW264.7 cells showing ROS
generation. RAW264.7 cells were infected with G37, MS5, MGRE or
treated with heat killed G37 (MOI 1:10) and generation of ROS was
detected by addition of DCF-DA. Images were captured using an
Olympus confocal laser scanning microscope with 488 nm laser. DCF-
DA and DIC indicate fluorescence of DCF-DA and differential
interference contrast of the same field; G37, MS5 and MGRE represent
cells infected with M. genitalium wild type, msrA mutant and control
strains. HKG37 represents cells treated with heat killed wild type M.
genitalium. t-BHP represents ROS induced with 1 mM t-butyl hydroper-
oxide for 30 min. B. Graphical representation of ROS generated
by RAW264.7 cells. Each bar represents ROS generated by RAW264.7
cells in response to infection/induction. Images were captured using a
confocal laser scanning microscope. Total fluorescence counts were
determined from images using NIH image J software from ten different
fields and three independent experiments. Arbitrary fluorescence units
for each infection are given as Mean 6 SD. *=p#0.05 vs wild type G37.
Labels are as described in ‘‘A’’.
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tiation of THP-1 cells may provide additional insights into this
mechanism. In M. fermentens, a 40 kDa surface lipoprotein has been
implicated in the differentiation of monocytes by this species .
Since M. genitalium has an array of surface lipoproteins that have
previously been implicated in induction of the host cell immune
response and alteration of host cell signaling [14,69,70], it is likely
that they also play a role in the differentiation of monocytes.
It is interesting to note that the msrA mutant strain of M.
genitalium is deficient in inducing the proinflammatory cytokine
TNF-a, the principal mediator of inflammatory response to
infection. A previous study has indicated that M. genitalium is
capable of inducing a variety of proinflammatory cytokines in
human monocyte derived macrophages , thus it is likely that
other proimflammatory cytokines may show similar responses to
this strain. Lipid associated membrane proteins (LAMPs) of M.
genitalium seems to be the major components that induce
proinflammatory cytokines in host cells [14,16,70] in which the
TLR2 receptor of the host cells play a major role . Blocking of
Figure 5. Differentiation of THP-1 cells by M. genitalium strains. A. Adherent THP-1 cells showing fluorescence. CFSE labeled THP-1 cells
were infected with M. genitalium strains (MOI 1:5). Images of adherent cells were acquired using confocal laser scanning microscope with 106
objective and 488 nm laser. G37, MS5 and MGRE are wild type, msrA mutant and control M. genitalium strains respectively. HKG37 represents heat
killed bacteria of wild type M. genitalium. B. Graph showing the amount of adherent cells for each infection. The number of labeled cells in
each image were counted using the particle plugin of Image J software. Average cell numbers from five different optical fields and from three
independent experiments were used for determining the number of adherent mononuclear cells in each infection. Labels are as described in ‘‘A’’.
*=p#0.05 vs wild type G37 strain.
Figure 6. Cytokines released by THP-1 cells after infection with mycoplasma strains. Supernatants from THP-1 cells infected with
mycoplasma strains (MOI 1:10) were collected and IL-1b and TNF-a concentrations were determined using an ELISA kit from eBioscience. A. Release
of IL-1b by THP-1 cells and B. Release of TNF-a by THP-1 cells. PBS, phosphate buffered saline control; G37 and MS5 are wild type msrA mutant M.
genitalium strains respectively; HKG37 represents heat killed wild type M. genitalium. *=p#0.05 vs wild type G37.
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the TLR2 receptors resulted in reduced induction of TNF-a and
IL-6 by LAMPs . It has been suggested that pathogenic
mycoplasmas adapt to different conditions in the host cells by
changing the size and phase variations of LAMPs .
Our findings show that MsrA in M. genitalium is primarily
localized in the cytoplasm. In contrast, some pathogenic bacteria
like A. actinomycetemcomitans , H. pylori  and N. gonorrhoeae
 have MsrA distributed between the cytosol and membrane,
although the proportions vary. Among these species, only A.
actinomycetemcomitans expresses MsrA as a single unit . In H.
pylori and N. gonorrhoeae MsrA is fused with either MsrB (MsrA-
MsrB), or with Trx and MsrB (Trx-MsrRA-MsrB) . It has
been reported that only the cytosolic MsrA was active in A.
actinomycetemcomitans  and only the membrane bound MsrA was
active in H. pylori . However, MsrA is a secretory protein in A.
actinomycetemcomitans, H. pylori and N. gonorrhoeae [73,74,75]. The
secreted MsrA should provide some benefit for these bacteria
either in defending host derived ROS or in modulating the host
cell machinery by reducing oxidized methionine in essential
In conclusion, it appears that the effect of MsrA on the virulence
of M. genitalium is primarily due to modification of surface
molecules that mediate interaction with host cells. Identification
of the molecules affected by MsrA may provide important clues for
the development of novel drugs against this pathogen.
and membrane fractions. Cytosol and membrane fractions
from M. genitalium strains, SDS-PAGE analysis and Western
transfer were done as described in Materials and Methods section.
The separated and transferred proteins were stained with Ponceau
S. G37 and MS5 represent M. genitalium wild type and msrA mutant
strains, respectively. W, M and C indicate whole, membrane and
cytosol fractions of M. genitalium. Mr indicates molecular weight
markers. Numbers on the left represent the sizes (kDa) of the
SDS-PAGE analysis of M. genitalium Cytosol
All confocal images were generated in the Optical Imaging Core Facility
supported by the Regional Medical Research Division, Edinburg-RAHC.
We thank Dr. Duncun Krause, Department of Microbiology, University of
Georgia, Athens, GA, for kindly providing M. pneumoniae anti-EF-G
antiserum. Also we thank Dr. James Bullard, Department of Chemistry,
University of Texas Pan American, for reading the manuscript and for
providing constructive criticisms.
Conceived and designed the experiments: SD. Performed the experiments:
KD GG SS. Analyzed the data: KD. Contributed reagents/materials/
analysis tools: SM. Wrote the paper: SD.
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