Ureaplasma parvum lipoproteins, including MB
antigen, activate NF-kB through TLR1, TLR2 and
Takashi Shimizu, Yutaka Kida and Koichi Kuwano
Division of Microbiology, Department of Infectious Medicine, Kurume University School of Medicine,
67 Asahi-machi, Kurume 830-0011, Japan
Received 20 December 2007
Revised 25 January 2008
Accepted 6 February 2008
Ureaplasma species (Ureaplasma parvum and Ureaplasma urealyticum) are commonly isolated
pathogens from the female reproductive tract and are associated with perinatal diseases in
humans. Inappropriate induction of inflammatory responses may be involved in the occurrence of
such diseases; however, pathogenic agents that induce the inflammatory response have not been
identified in ureaplasmas. In this study, we examined the involvement of Toll-like receptors
(TLRs) in the activation of the immune response by U. parvum lipoproteins, as well as the U.
parvum components responsible for nuclear factor kB (NF-kB) activation. The Triton X-114
(TX-114) detergent phase of U. parvum was found to induce NF-kB through TLR2. The active
components of the TX-114 detergent phase were lipoproteins, such as multiple banded (MB)
antigen, UU012 and UU016 of U. parvum. The activation of NF-kB by these lipoproteins was
inhibited by dominant negative (DN) constructs of TLR1 and DN TLR6. Thus, the lipoproteins from
U. parvum were found to activate NF-kB through TLR1, TLR2 and TLR6. Furthermore, these
lipoproteins possessed an ability to induce tumour necrosis factor-alpha (TNF-a) in mouse
Ureaplasmas are wall-less bacteria belonging to the
(Ureaplasma parvum and Ureaplasma urealyticum) are
common commensals of the urogenital tract of humans,
ureaplasmas have frequently been recognized as important
(Shepard, 1954), chorioamnionitis (Cassell et al., 1986;
Maher et al., 1994), preterm birth (Jelsema, 2006; Kataoka
et al., 2006; Ollikainen et al., 1998), infertility (Taylor-
Robinson, 1986; Xu et al., 1997), pneumonia in neonates
(Waites et al., 1989) and bronchopulmonary dysplasia in
neonates (Schelonka et al., 2005). The mechanisms by
which ureaplasmas cause such diseases are unclear, but the
inappropriate induction of inflammatory responses may be
involved. It has been reported that the levels of interleukin-
6 (IL-6), tumour necrosis factor-alpha (TNF-a), IL-1b and
IL-8 in intra-amniotic fluid infected with ureaplasmas are
increased (Yoon et al., 1998). Moreover, macrophages are
capable of producing such cytokines and nitric oxide in
response to U. urealyticum (Li et al., 2000a, b). However,
pathogenic agents such as endotoxin and exotoxin that
induce an inflammatory response have not been identified
It has been widely known that Toll-like receptors (TLRs)
with the function of pattern-recognition receptors play
critical roles in early innate recognition and inflammatory
responses by the host against invading microbes (Akira &
Takeda, 2004; Kopp & Medzhitov, 1999). Among 10 TLR
family members reported, TLR2, TLR4, TLR5 and TLR9
have been implicated in the recognition of different
bacterial components. Peptidoglycan (PGN), lipoarabino-
mannan, zymosan and lipoproteins from various micro-
organisms are recognized by TLR2 (Aliprantis et al., 1999;
Brightbill et al., 1999; Lien et al., 1999; Means et al., 1999;
Takeuchi et al., 1999, 2000, 2002; Underhill et al., 1999).
On the other hand, LPS, bacterial flagellin and bacterial
DNA are recognized by TLR4, TLR5 and TLR9, respect-
ively (Hayashi et al., 2001; Hemmi et al., 2000; Hoshino
et al., 1999; Poltorak et al., 1998). These TLR family
members have been shown to activate nuclear factor
kB (NF-kB) via IL-1R-associated
including myeloid differentiation protein (MyD88), IL-
1R-activated kinase (IRAK), TNFR-associated factor 6
Abbreviations: DN, dominant negative; FAM20, synthetic lipopeptide
containing N-terminal 20 amino acids of F0F1-type ATPase derived from
Mycoplasma pneumoniae; IL, interleukin; MALP-2, macrophage-activ-
ating lipopeptide 2; MB, multiple banded; NF-kB, nuclear factor kB;
Cys-(S)-Ser-(S)-Lys4-OH, 3HCl; PGN, peptidoglycan; PMF, peptide
mass fingerprinting; sN-ALP2, synthesized NF-kB-activating lipopeptide
2; TLR, Toll-like receptor; TNF-a, tumour necrosis factor-alpha; TX-114,
Microbiology (2008), 154, 1318–1325
1318 2007/016212G2008 SGM Printed in Great Britain
(TRAF6) and NF-kB-inducing kinase (NIK) (Medzhitov
et al., 1998).
In this study, we examined the involvement of TLRs in the
activation of the immune response by lipoproteins from U.
parvum, as well as the U. parvum components responsible
for NF-kB activation. We observed that the Triton X-114
(TX-114) detergent phase of U. parvum could induce NF-
kB through TLR2. The active components of the TX-114
detergent phase responsible for NF-kB activation were
found to be 75 and 55 kDa proteins (P75 and P55,
respectively). The NF-kB-inducing activity of P75 was
much higher than that of P55. By analysis with peptide
mass fingerprinting (PMF), P75 was matched to a multiple
banded (MB) antigen of U. parvum, and P55 was found to
be a mixture of lipoproteins containing UU012 and
UU016. The activation of TLR signalling by P75 and P55
was apparently dependent on TLR1, TLR2 and TLR6.
These lipoproteins also induced TNF-a in mouse periton-
eal macrophages. Thus, the results indicate that the MB
antigen induces inflammatory responses that might be
responsible for pathogenesis in U. parvum infection.
Therefore, the MB antigen might be a candidate molecule
for the prevention and therapy of U. parvum infection.
Cells. Cells of a human kidney cell line, 293T, were cultured in
Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal
calf serum (FCS), 2 mM L-glutamine, 100 U penicillin G ml21and
100 mg streptomycin ml21.
TX-114 phase partitioning. U. parvum serovar 3 (ATCC 700970)
was cultured in PPLO broth, pH 6.0, containing 20% horse serum,
0.25% yeast extract and 100 mg ampicillin ml21to the beginning of
stationary phase, and then pelleted by centrifugation for 30 min at
12000 g. TX-114 phase partitioning was performed as described by
Feng & Lo (1994, 1999). Briefly, a ureaplasma pellet was suspended in
Tris-buffered saline (TBS) containing 1 mM EDTA (TBSE), solubi-
lized by adding TX-114 to a final concentration of 2%, and incubated
at 4 uC for 1 h. The lysate was incubated at 37 uC for 10 min for
phase separation. After centrifugation at 10000 g for 20 min, the
upper aqueous phase was removed and replaced by the same volume
of TBSE. The procedure of phase separation was repeated twice. The
final TX-114 detergent phase was resuspended in TBSE to the original
volume, and 2.5 volumes of ethanol were added to precipitate
membrane components, followed by incubation at 220 uC overnight.
After centrifugation, the pellet was suspended in DMSO. The aqueous
phase was washed two times by adding TX-114 to a final
concentration of 2%. The TX-114-insoluble pellet was dissolved in
DMSO. The protein concentration of the suspension was measured
by Protein Assay CBB Solution (Nacalai Tesque).
Expression vectors. To prepare TLR1, TLR2 and TLR6 expression
vectors (pFLAG-TLR1, pFLAG-TLR2 and pFLAG-TLR6), the coding
regions of TLR1, TLR2 and TLR6 minus the respective N-terminal
signal sequences were amplified by PCR from a cDNA of THP-1 and
cloned into the expression vector pFLAG-CMV1 (Sigma), in which a
Dominant negative (DN) TLR1 and TLR6 expression vectors were
constructed by subcloning Toll and IL-1 receptor (TIR) homology
domain-deleted TLR1 and TLR6 fragments into pFLAG-CMV1
(pFLAG-dTLR1 and pFLAG-dTLR6, respectively). The NF-kB
an N-terminalFLAG epitope.
cis-Reporting System containing pNF-kB-luc was purchased from
Transfection and luciferase assay. Transient transfection was
performed by using FuGENE 6 (Roche) according to the manufac-
turer’s instructions. A total of 16105293T cells were transfected with
0.1 mg pFLAG-TLR2, 0.01 mg pNF-kB-luc, 0.01 mg of pRL-TK
internal control plasmid (Promega) and 0.2 mg of DN TLRs
expressing plasmid in 24-well plates. After 48 h, transfected cells
were stimulated with TX-114 detergent phase or purified proteins.
After a further 8 h incubation, cells were lysed and assayed for
luciferase activity using a Dual-Luciferase Reporter Assay System
(Promega). Firefly and Renilla luciferase activity was monitored with
a Lumat LB 9507 luminometer (Berthold). Normalized reporter
activity was expressed as the firefly luciferase value divided by the
Renilla luciferase value. Relative fold induction was calculated as the
normalized reporter activity of the test samples divided by that of
the unstimulated samples.
Purification from acrylamide gel. One hundred micrograms of
TX-114 detergent phase was separated by 7.5% glycine- SDS-PAGE
gels under reducing conditions. The gels were stained with Quick
CBB Plus (Wako). To elute proteins, the stained gel was cut into
5 mm strips and each strip was homogenized with 1% SDS solution.
The homogenized gel was removed by centrifugation at 12000 g at
room temperature. Five volumes of acetone were added to the
supernatant, followed by incubation overnight. Proteins were pelleted
by centrifugation at 12000 g and 4 uC, and dissolved in 100 ml
DMSO. The protein concentration of the suspension was measured
by Protein Assay CBB Solution (Nacalai Tesque).
PMF. PMF was carried out according to the method of Dr K. Yoshino
(Yoshino et al., 2004). Briefly, the Coomassie Brilliant Blue (CBB)- or
silver-stained bands obtained from Tricine-SDS-PAGE were excised
and sliced into small strips. To remove CBB, the strips were incubated
in 50% methanol/5% acetic acid for 1 h and washed two times in
water. The strips were dehydrated by incubation with 100%
acetonitrile. To alkylate the protein, the strips were incubated at
60 uC for 1 h with 10 mM DTT in 100 mM ammonium hydrogen
carbonate, followed by treatment at room temperature for 30 min
with 55 mM iodoacetamide (Nacalai Tesque) in 100 mM ammonium
hydrogen carbonate. In-gel trypsin digestion was carried out by
incubation with 10 mg trypsin ml21(Promega). The digested peptides
were eluted by 5% formic acid (Wako). Peptides were dried in vacuo
and dissolved in saturated a-cyano-4-hydroxycinnamic acid (Nacalai
Tesque) in 50% acetonitrile and 0.1% trifluoroacetic acid. The
molecular masses of the peptides were measured with an Autoflex
MALDI-TOF mass spectrometer (Bruker Daltonics). The SWISS-
PROT database was searched by MASCOT (Science Matrix).
Macrophage stimulation. To induce peritoneal macrophages,
0.1 mg of OK432 (Chugai Pharmaceutical) was injected into the
peritoneal cavity of C57BL mice. Two days later, peritoneal exudate
cells as macrophages were harvested and centrifuged. The cell pellets
were suspended in serum-free medium (SFM) optimized for
macrophage culture (Gibco Invitrogen). Cells were allowed to adhere
to 96-well culture plates for 2 h at 37 uC with 5% CO2. Non-adherent
cells were removed by washing with PBS, and the remaining adherent
cells were stimulated with TX-114 detergent phase or purified
proteins for 6 h. The production of TNF-a was measured using an
ELISA kit (R&D systems).
Statistical analysis. Results expressed as means and SD were
compared using one-way ANOVA. The differences between each
group were compared by multiple comparisons (Bonferroni t test).
Differences were considered significant at P,0.05.
NF-kB activation by lipoproteins from U. parvum
TLR2-dependent activation of NF-kB by the TX-
114 detergent phase of U. parvum
Ureaplasmas are wall-less bacteria, and completely lack LPS
and PGN, which are ligands for TLR4 and TLR2,
respectively (Weisburg et al., 1989). U. parvum has no
flagella, the ligand for TLR5 (Glass et al., 2000). Other than
these components, therefore, lipoproteins, the ligands for
TLR2, are possible candidates for an inflammation-
inducing factor in U. parvum. In fact, we previously
demonstrated that lipoproteins derived from Mycoplasma
pneumoniae induce NF-kB through TLR2 (Shimizu et al.,
2005). To elucidate whether U. parvum induces NF-kB
through TLR2, we initially performed TX-114 phase
partitioning of U. parvum. 293T cells were transfected
with both a TLR2 expression vector pFLAG-TLR2 and a
reporter vector pNF-kB-luc, in which the luciferase
reporter gene was fused to an NF-kB enhancer. When
293T cells transfected with pFLAG-TLR2 were stimulated
with the TX-114-insoluble phase and the aqueous phase,
the levels of luciferase expression were slightly increased
(Fig. 1). In contrast, when the cells were stimulated with
the TX-114 detergent phase of U. parvum, the expression
level was remarkably augmented, and the activity was
approximately fivefold higher than those of the TX-114-
insoluble phase and the aqueous phase. Upon stimulation
with culture medium, the level of luciferase expression was
the same as the unstimulated control level. When 293T
cells were transfected with an empty vector pFLAG-CMV1,
the level of luciferase expression was not augmented
(Fig. 1). The results suggest that the TX-114 detergent
phase of U. parvum induces NF-kB activation through
Purification of NF-kB-activating components of
the TX-114 detergent phase
To examine the active components of the TX-114 detergent
phase, the TX-114 detergent phase was separated by
glycine-SDS-PAGE, and the gel was excised into 10 pieces
(Fig. 2a). Proteins extracted from each gel piece as
described in Methods were incubated with 293T cells
transfected with pFLAG-TLR2 and pNF-kB-luc. When the
cells were incubated with the proteins extracted from the
gel strips, 75 and 55 kDa proteins (P75 and P55,
respectively) were found to possess NF-kB-inducing
activity (Fig. 2b). The NF-kB-inducing activity of P75
was approximately three times higher than that of P55.
Identification of 75 kDa NF-kB-activating
First, PMF analysis for P75 was carried out, since the
protein was abundant and showed a relatively strong NF-
kB-inducing activity. As the result of a database search of
SWISS-PROT using MASCOT, P75 was matched to the
Fig. 1. TX-114 phase partitioning. 293T cells were transfected
with 0.01 mg pFLAG-TLR2 ml”1, 0.01 mg pNF-kB-luc ml”1and
0.01 mg pRL-TK ml”1. The cells were stimulated with 1.0 mg ml”1
culture medium, TX-114-insoluble phase, aqueous phase and TX-
114 detergent phase of U. parvum. All values represent the means
and SDs of three assays. **P,0.01 versus PBS.
Fig. 2. Molecular masses of the active com-
ponents. (a) TX-114 detergent phase was
separated by 7.5% glycine-SDS-PAGE gels
under reducing conditions. Ten major protein
bands were elicited from the gels and the
proteins were extracted. (b) Eluted solutions
were incubated with 293T cells transfected
with0.01 mg pNF-kB-luc
pFLAG-TLR2 ml”1and 0.01 mg pRL-TK ml”1
at final concentrations
Luciferase activity was measured as described
in Methods. All values represent the means
and SDs of three assays. *P,0.05 versus PBS;
**P,0.01 versus control.
ml”1, 0.01 mg
of100 ng ml”1.
T. Shimizu, Y. Kida and K. Kuwano
MB antigen of U. parvum (Fig. 3a). The protein score for
MB antigen was 102; scores greater than 77 are considered
to be significant (P,0.05). MB antigen contains a variable
number of repeat sequences and hence shows different
molecular masses in different isolates. In U. parvum serovar
3, MB antigen contains 42 repeats. The estimated
molecular masses are 42845 and 39830 Da for the native
and processed forms, respectively; however, MB antigen
migrates at ~75 kDa by SDS-PAGE (Zheng et al., 1995).
MB antigen contains a signal peptide near the N-terminal
end followed by a predicted lipid-binding cysteine. These
findings indicate that MB antigen should be a lipoprotein
(Zheng et al., 1995).
Cooperation of TLR2 and TLR6 for NF-kB
Diacylated lipopeptides, including macrophage-activating
lipopeptide 2 (MALP-2), have been reported to be
recognized by mouse TLR2 cooperatively with TLR6
(Takeuchi et al., 2001). To investigate whether MB antigen
is also recognized by both TLR2 and TLR6 for NF-kB
activation, we transfected a plasmid encoding DN TLR6
(pFLAG-dTLR6) into 293T cells with both pFLAG-TLR2
and pNF-kB-luc. Initially, the levels of expression of TLR1
and TLR6 in 293T cells were analysed by RT-PCR. Both
TLR1 and TLR6 were found to be transcribed in 293T
cells (data not shown). The effect of DN TLR6 on the
expression of TLR2 was also analysed by flow cytometry.
The level of TLR2 expression was almost constant,
irrespective of the expression of DN TLR6 or DN TLR1
(data not shown). When the cells transfected with control
vector were stimulated with P75, the levels of NF-kB
activation were increased (Fig. 4). When the cells were
transfected with DN TLR6, the levels of NF-kB activation
were strikingly decreased. These results suggest that NF-kB
activation by P75 is dependent on TLR6. As a control,
synthetic lipopeptides derived from M. pneumoniae
(Shimizu et al., 2008) were used in this assay. When
DN TLR6-transfected cells were stimulated with a synthetic
diacylated lipopeptide, FAM20 (synthetic lipopeptide
containing N-terminal 20 amino acids of F0F1-type
ATPase derived from Mycoplasma pneumoniae), the
level of NF-kB activation was decreased by DN TLR6.
Meanwhile, when the cells were stimulated with a synthetic
triacylated lipopeptide sN-ALP2 (synthesized NF-kB-activ-
ating lipopeptide 2), NF-kB activation was unaffected by
DN TLR6 (Fig. 4).
Fig. 3. PMF. Amino acid sequences of MB antigen (a), UU012 (b)
and UU016 (c). Box, signal peptide; *lipid binding cysteine;
underlined type, mature protein; bold type, peptides detected by
Fig. 4. Cooperation of TLR1, TLR6 and TLR2 for NF-kB induction
by MB antigen. 293T cells were transfected with 0.2 mg ml”1
pFLAG-dTLR6 or pFLAG-dTLR1, 0.01 mg pFLAG-TLR2 ml”1,
0.01 mg pNF-kB-luc ml”1and 0.01 mg pRL-TK ml”1. The cells
were stimulated with 10 ng P75 ml”1, 100 ng P55 ml”1, 10 ng
FAM20 ml”1or 100 ng sN-ALP1 ml”1. All values represent the
means and SDs of three assays. **P,0.01 versus vector.
NF-kB activation by lipoproteins from U. parvum
Cooperation of TLR1 and TLR2 for NF-kB
Triacylated bacterial lipopeptides such as Pam3CSK4have
been reported to be recognized by murine TLR1 in
association with TLR2 (Takeuchi et al., 2002). We next
determined whether P75 is recognized by both TLR1 and
TLR2 for NF-kB activation. We transfected a plasmid
encoding DN TLR1 (pFLAG-dTLR1) into 293T cells with
both pFLAG-TLR2 and pNF-kB-luc. When the cells were
transfected with control vector, the stimulation with P75
augmented the levels of NF-kB induction compared to
background levels. In contrast, upon transfection with DN
TLR1, the NF-kB activation by P75 was significantly
decreased (Fig. 4). As for the control, the NF-kB activation
by the diacylated lipopeptide (FAM20) and the triacylated
lipopeptide (sN-ALP2) was decreased by DN TLR1 (Fig. 4).
These results indicate that the cooperation of TLR1 is
required for the NF-kB activation by P75.
Identification of 55 kDa NF-kB-activating
We next tried to identify P55. The results of PMF showed
that P55 was a mixture of lipoproteins UU012 and UU016
of U. parvum (Fig. 3b, c). The protein scores for UU012
and UU016 were 89 and 113, respectively; scores greater
than 77 are considered to be significant (P,0.05). It was
technically difficult to separate UU012 and UU016 because
of their similar molecular masses and isoelectric points.
The estimated molecular mass of UU012 is 56682 and
53735 Da for the native and processed form, respectively.
Similarly, that of UU016 is 57241 and 54198 Da for the
native and processed form, respectively. Both UU012 and
UU016 contain a signal peptide near the N-terminal end
followed by a predicted lipid-binding cysteine. These
findings indicate that UU012 and UU016 are also
lipoproteins (Glass et al., 2000).
Cooperation of TLR1, TLR2 and TLR6 in NF-kB
activation by P55
We next determined whether P55 is dependent on TLR1 or
TLR6 to induce NF-kB activation. We transfected a plasmid
encoding DN TLR1 and DN TLR6 into 293T cells with both
pFLAG-TLR2 and pNF-kB-luc. When the cells were
transfected with the control vector, stimulation with P55
augmented the levels of NF-kB activation. In contrast, upon
transfectionwith DNTLR1 orDNTLR6,thelevelsofNF-kB
activation were significantly decreased compared with the
control (Fig. 4). These results indicate that NF-kB activation
by P55 is also dependent on TLR1 and TLR6.
Induction of TNF-a by TX-114 detergent phase,
and by P75 and P55
To investigate whether these components derived from
U. parvum actually induce inflammatory cytokines in
macrophages, the levels of TNF-a production by mouse
peritoneal macrophages stimulated with TX-114 detergent
phase, P75 or P55 were measured by ELISA. Peritoneal
macrophages treated with PBS failed to produce TNF-a.
On the other hand, the levels of TNF-a production were
augmented by TX-114 detergent phase, P75 and P55
(Fig. 5). These results suggest that TX-114 detergent phase,
P75 and P55 possess the ability to induce inflammatory
cytokines, including TNF-a.
In this study, we demonstrated that lipoproteins from U.
parvum activate NF-kB through TLR1-, TLR2- and TLR6-
dependent pathways in 293T cells, and induce TNF-a in
mouse peritoneal macrophages. The lipoproteins were
found to be MB antigen, UU012 and UU016 of U. parvum.
In this study, it was difficult to separate UU012 and
UU016, since they are very close in molecular mass (53735
and 54198 Da, respectively) and isoelectric point (8.6 and
8.4, respectively). In the current study, we did not
determine which protein of UU012 and UU016 has the
NF-kB-inducing activity. However, both proteins are
assumed to be lipoproteins with the ability to induce NF-
kB activation based on their amino acid sequences.
Interestingly, MB antigen was abundant in the TX-114
detergent phase, and its ability to induce NF-kB activation
was higher than that of the mixture of UU012 and UU016.
These results indicate that MB antigen might be a key
factor to induce inflammatory responses.
It is known that triacylated lipoproteins are recognized by
TLR1 and TLR2, whereas diacylated lipoproteins are
recognized by TLR2 and TLR6. In TLR1-deficient mice,
Fig. 5. TNF-a induction by TX-114 detergent phase, P75 and
P55. A total of 3?105peritoneal macrophages per well obtained
from C57BL mice were stimulated with 1.0 mg ml”1of TX-114
detergent phase, 10 ng P75 ml”1or 10 ng P55 ml”1in a 96-well
plate for 6 h. Levels of TNF-a in the culture supernatants were
measured using ELISA kits. All values represent the means and
SDs of three independent experiments. **P,0.01 versus PBS.
T. Shimizu, Y. Kida and K. Kuwano
Pam3CSK4 containing three acyl chains fails to induce
cytokines, including TNF-a, while MALP-2 containing two
acyl chains can induce cytokines (Takeuchi et al., 2002). In
TLR6-deficient mice, MALP-2 fails to induce cytokines,
but Pam3CSK4can induce them (Takeuchi et al., 2001). In
this study, MB antigen and the mixture of UU012 and
UU016 induced NF-kB activation through TLR1-, TLR2-
and TLR6-dependent pathways. Curiously, it appears to be
unclear whether mycoplasmas and ureaplasmas have
triacylated lipoproteins. In triacylated lipoproteins, three
fatty acids are bound to the N-terminal cysteine residue:
two in a diacylglyceride that is linked through a thioether
bond to the thiol group and one to the amine group
(N-acylation). Chemically identified lipoproteins from
Mycoplasma fermentans, Mycoplasma hyorhinis, Myco-
plasma salivarium and Mycoplasma gallisepticum are not
N-acylated (Jan et al., 1996a, 2001; Muhlradt et al., 1997,
1998; Shibata et al., 2000). An N-acyltransferase gene
responsible for N-acylation has not been detected in the M.
pneumoniae, Mycoplasma genitalium, Mycoplasma pene-
trans and U. parvum genomes (Fraser et al., 1995; Glass
et al., 2000; Himmelreich et al., 1996; Sasaki et al., 2002)
either. However, a study of the ratio of N-amide and O-
ester bonds in M. gallisepticum and Mycoplasma mycoides
suggests that such mycoplasmas possess diacylated and
triacylated lipoproteins (Jan et al., 1996b). Furthermore,
the resistance to Edman degradation of proteins from M.
mycoides also indicates the presence of N-acylation
(Chambaud et al., 1999). We previously tested the TLR
dependency of synthetic diacylated or triacylated lipopep-
tides derived from M. pneumoniae. The synthetic triacy-
lated lipopeptides, such as sN-ALP2, induce NF-kB
activation through TLR1 and TLR2, whereas diacylated
lipopeptides, such as FAM20, induce through TLR1, TLR2
and TLR6 (Shimizu et al., 2007, 2008) (Fig. 4). These
results indicate that MB antigen, UU012 and UU016 may
be diacylated lipoproteins.
Ureaplasmas are known to be associated with non-
gonococcal urethritis (Shepard, 1954), chorioamnionitis
(Cassell et al., 1986; Maher et al., 1994), preterm birth
(Jelsema, 2006; Kataoka et al., 2006; Ollikainen et al.,
1998), infertility (Taylor-Robinson, 1986; Xu et al., 1997),
pneumonia in neonates (Waites et al., 1989) and
bronchopulmonary dysplasia in neonates (Schelonka et al.,
2005). The urease and phospholipase produced by
ureaplasmas have been implicated as virulence factors
(De Silva & Quinn, 1986; Ligon & Kenny, 1991). In
responses may be involved in the development of these
diseases (Yoon et al., 1998). In this study, we identified
lipoproteins, such as MB antigen, UU012 and UU016, that
can induce inflammatory responses. The functions of MB
antigen are unknown, but it has been reported to be a
major component of U. parvum and has been detected in
various serovars (Watson et al., 1990; Zheng et al., 1992).
Since there is an association between the amino acid
sequences of lipoproteins and TLR dependency and
stimulatory activity (Buwitt-Beckmann et al., 2005), the
size variation of MB antigen in different serovars may affect
the TLR dependency and stimulatory activity. To our
knowledge, this study is the first report of a virulence factor
of U. parvum that induces an inflammatory response.
However, further study is needed to clarify the in vivo
activity of the lipoproteins.
Considering the interaction between TLR and lipoproteins
such as MB antigen, the inhibition of the interaction may
result in the reduction of the inflammatory response
induced by U. parvum. Accordingly, our data suggest that
such lipoproteins are potential therapeutic targets. The
lipoproteins and their derivatives might be suitable
candidates as antigens for vaccination, causing the
induction of a specific antibody capable of inhibiting
TLR signalling. Alternatively, the derivatives may work as
antagonists that inhibit TLR signalling. Furthermore,
several reports indicate that mycoplasma-derived lipopro-
teins possess immune modulation and adjuvant activities
(Link et al., 2004; Rharbaoui et al., 2002, 2004; Romero
et al., 2004). Thus, lipoproteins capable of interacting with
TLR might become potential molecules in the development
of new therapeutics against U. parvum infection.
We thank Dr T. Ishikawa (Fukuoka Industrial Technology Center) for
MS analysis. This work was supported in part by a Grant-in-Aid from
the Ministry of Education, Science, Sports and Culture of Japan.
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Edited by: J. Renaudin
NF-kB activation by lipoproteins from U. parvum