Pam3Cys (OspA) activates HIV replication
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Jul 21, 2008, 2:51:19 PM7/21/08
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Subject: Pam3Cys (OspA) activates HIV replication
Date: Jul 21, 2008 8:45 AM
That's a considerable number of research-years and lives lost to the
lies
about LYMErix and Lyme Disease.
Imagine if Yale told the truth instead of blew these people off
deliberately:
http://www.actionlyme.org/SCHOEN_INSTRUCTING_DOCS_TO_BLOW_OFF_LYMERIX_INJUREES.htm
http://www.actionlyme.org/Bull_Lewis.htm
Your tax dollars at work.
http://www.actionlyme.org/BIOMARKERS2.htm
Paul Duray told these bastards about it in 1992.
KMDickson
http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=15312143
Immunology. 2004 September; 113(1): 121–129.
doi: 10.1111/j.1365-2567.2004.01937.x.
PMCID: PMC1782549
Copyright © 2004 Blackwell Publishing Ltd
Lipid-associated membrane proteins of Mycoplasma fermentans and M.
penetrans activate
human immunodeficiency virus long-terminal repeats through Toll-like
receptors
Takashi Shimizu, Yutaka Kida, and Koichi Kuwano
Department of Bacteriology, Kurume University School of Medicine,
Kurume, Japan
K. Kuwano, 67 Asahi-machi, Kurume, Fukuoka 830-0011, Japan. E-mail:
kuw...@med.kurume-u.ac.jp
Received April 21, 2004; Revised May 25, 2004; Accepted June 2, 2004.
Small right arrow pointing to: This article has been cited by other
articles in
PMC.
Top
>Abstract
Introduction
Materials and methods
Results
Discussion
References
Abstract
Mycoplasmas are known to enhance human immunodeficiency virus (HIV)
replication,
and mycoplasma-derived lipid extracts have been reported to activate
nuclear factor-κB
(NF-κB) through Toll-like receptors (TLRs). In this study, we examined
the involvement
of TLRs in the activation of HIV long-terminal repeats (LTR) by
mycoplasma and their
active components responsible for the TLR activation. Lipid-associated
membrane
proteins (LAMPs) from two species of mycoplasma (Mycoplasma fermentans
and M. penetrans)
that are associated with acquired immune-deficiency syndrome (AIDS),
were found
to activate HIV LTRs in a human monocytic cell line, THP-1. NF-κB
deletion from
the LTR resulted in inhibition of the activation. The LTR activation
by M. fermentans
LAMPs was inhibited by a dominant negative (DN) construct of TLR1 and
TLR6, whereas
HIV LTR activation by M. penetrans LAMPs was inhibited by DN TLR1, but
not by DN
TLR6. These results indicate that the activation of HIV LTRs by M.
fermentans and
M. penetrans LAMPs is dependent on NF-κB, and that the activation of
HIV LTR by
M. fermentans LAMPs is mediated through TLR1, TLR2 and TLR6. In
contrast, the LTR
activation by M. penetrans LAMPs is carried out through TLR1 and TLR2,
but not TLR6.
Subsequently, the active component of M. penetrans and M. fermentans
LAMPs was purified
by reverse-phase high-performance liquid chromatography (HPLC).
Interestingly, the
purified lipoprotein of M. penetrans LAMPs (LPMp) was able to activate
NF-κB through
TLR1 and TLR2. On the other hand, the activation of NF-κB by purified
lipoprotein
of M. fermentans LAMPs (LPMf) was mediated through TLR2 and TLR6, but
not TLR1.
Keywords: HIV, lipoprotein, mycoplasma, Toll-like receptor
Top
Abstract
>Introduction
Materials and methods
Results
Discussion
References
Introduction
Human immunodeficiency virus (HIV) is recognized as the aetiological
agent of acquired
immune-deficiency syndrome (AIDS). However, the progression of AIDS is
highly variable
in different individuals, and several factors, such as viral strains
or host factors,
have been attributed as the possible cause of such variations.
Infectious agents,
including various viruses, parasites and bacteria, are considered to
be cofactors
in the progression of AIDS.1 Mycoplasmas are wall-less parasitic Gram-
positive bacteria,
and the smallest organisms capable of self-replication.2Mycoplasma
fermentans and
M. penetrans have been isolated from the tissues and urine of patients
with AIDS3–5
and were shown to enhance the cytopathic effect of HIV-1 infection.6,7
In addition,
mycoplasmas and acholeplasmas have been reported to enhance HIV-1
replication in
vitro.8,9 Thus, mycoplasmas might be reasonable cofactor candidates in
the progression
of AIDS.
A nuclear transcription factor – nuclear factor-κB (NF-κB) – is
thought to play
a major role in the regulation of HIV-1 gene expression.10 Although
the HIV long-terminal
repeat (LTR) alone can serve as its own promoter, early mRNA
transcription appears
to rely primarily on the binding of cellular transcription factors,
including NF-κB,
to the LTR.11 The activation of cytoplasmic NF-κB by various
cytokines, including
interleukin (IL)-2, IL-6 and tumour necrosis factor-α (TNF-α), or
after infection
with other viruses, induces HIV replication.12–17 These findings
indicate increased
rates of HIV replication, probably through NF-κB-mediated regulation
of the HIV
LTR. TNF-α induced by mycoplasma had been thought to induce NF-κB and
HIV replication;
however, anti-TNF-α immunoglobulin failed to inhibit the enhancement
of HIV replication
by mycoplasma.18 Although activation of NF-κB has been implicated in
the mycoplasma-induced
enhancement of HIV replication, the receptor(s) and the pathways of
signal transduction
via NF-κB have not been clearly defined.
Recently, it has been reported that Toll-like receptors (TLRs) are
pattern-recognition
receptors in the innate immune system and play important roles in
early innate recognition
and inflammatory responses by the host to microbial challenges.19
Among nine TLR
family members reported, TLR2, 4, 5 and 9 have been implicated in the
recognition
of different bacterial components. Pepidoglycan, lipoarabinomannan,
zymosan and
lipoproteins from various micro-organisms are recognized by TLR2.20–28
On the other
hand, lipopolysaccharide (LPS), bacterial flagellin and bacterial DNA
are recognized
by TLR4, TLR5 and TLR9, respectively.29–33 These TLR family members
have been shown
to activate NF-κB via IL-1R-associated signal molecules, including
myeloid differentiation
protein (MyD88), IL-1R-activated kinase (IRAK), TNFR-associated factor
6 (TRAF6),
and NF-κB-inducing kinase (NIK).34 However, the precise mechanisms by
which mycoplasma
activate HIV LTR have not been fully clarified.
In this study, we examined the involvement of TLRs in the activation
of HIV LTRs
by mycoplasmas and their active components responsible for the TLR
activation. We
observed that lipid-associated membrane proteins (LAMPs) from the AIDS-
associated
mycoplasmas, M. penetrans and M. fermentans, activated the HIV LTR in
a human monocytic
cell line (THP-1) through NF-κB. Activation of the HIV LTR by LAMPs
from M. fermentans
(referred to as M. fermentans LAMPs hereafter) was apparently
dependent on TLR1,
TLR2 and TLR6. In contrast, activation of the HIV LTR by LAMPs from M.
penetrans
(referred to as M. penetrans LAMPs hereafter) was dependent on TLR1
and TLR2, but
not on TLR6. Furthermore, the active components of M. penetrans and M.
fermentans
LAMPs were purified by reverse-phase HPLC. The activity of purified
lipoprotein
from M. penetrans LAMPs (LPMp) to induce NF-κB was dependent on TLR1
and TLR2. On
the other hand, the activity of purified lipoprotein from M.
fermentans LAMPs (LPMf)
was dependent on TLR2 and TLR6, but not on TLR1.
Top
Abstract
Introduction
>Materials and methods
Results
Discussion
References
Materials and methods
Cells
Cells of a human monocytic cell line, THP-1, were cultured in
RPMI-1640 containing
10% fetal calf serum (FCS; Mitsubishi Chemical, Tokyo, Japan), 2 mm l-
glutamine,
100 U/ml penicillin G and 100 µg/ml streptomycin. Cells of a human
kidney cell line,
293T, were cultured in Dulbecco's modified Eagle's minimal essential
medium
(DMEM) containing 10% FCS, 2 mm l-glutamine, 100 U/ml penicillin G and
100 µg/ml
streptomycin.
Antibodies
The mouse anti-human TLR2 monoclonal antibodies (mAbs) ABM-8320 and
IMG-416 were
obtained from Cascade Bioscience (Winchester, MA) and Imgenex (San
Diego, CA).35
Normal mouse immunoglobulin G (IgG)2a was purchased from PharMingen
(San Diego,
CA).
Pathogen-associated molecular patterns (PAMPs)
(S)-[2,3-Bis(palmitoyloxy)-(2-RS)-propyl]-N-palmitoyl-(R)-Cys-(S)-Ser-
(S)-Lys4-OH.3HCl
(Pam3CSK4) was purchased from Calbiochem (Darmstadt, Germany). M.
fermentans macrophage-activating
lipopeptide 2 (MALP-2) was kindly provided by Dr M. Matsumoto (Osaka
Medical Center
for Cancer and Cardiovascular Diseases, Osaka, Japan).36,37
Preparation of LAMPs from M. fermentans and M. penetrans
M. fermentans and M. penetrans were cultured in PPLO medium and SP-4
medium, respectively,
to the start of stationary phase, and then pelleted by centrifugation
for 10 min
at 12 000 g. Preparation of LAMPs was performed as described
previously by Feng
et al.38,39 Briefly, a mycoplasma pellet was suspended in Tris-
buffered saline (TBS)
(50 mm Tris, 0·15 m NaCl, pH 8·0) containing 1 mm EDTA (TBSE),
solubilized by adding
TX-114 to a final concentration of 2% and incubated at 4° for 1 hr.
The lysate was
incubated at 37° for 10 min prior to phase separation. After
centrifugation at 10
000 g for 20 min, the upper aqueous phase was removed and replaced
with the same
volume of TBSE. The procedure of phase separation was repeated twice.
The final
TX-114 phase was resuspended in TBSE to the original volume, 2·5
volumes of ethanol
were added to precipitate membrane components and the phase was
incubated at −20°
overnight. After centrifugation, the pellet was suspended in phosphate-
buffered
saline (PBS) followed by sonication for 30 seconds at output 5
(Sonifier cell disruptor
200; Branson, Danbury, CT). The protein concentration of the
suspension was measured
by using the Coomassie Protein Assay Regent (Pierce, Rockford, IL).
Expression vectors
To prepare TLR1, TLR2 and TLR6 expression vectors (pFLAG-TLR1, pFLAG-
TLR2, and pFLAG-TLR6,
respectively), the coding regions of TLR1, TLR2 and TLR6, minus the
respective N-terminal
signal sequences, were amplified by polymerase chain reaction (PCR)
from a cDNA
of THP-1 and cloned into the expression vector pFLAG-CMV1 (Sigma, St
Louis, MO),
in which a preprotrypsin leader precedes an N-terminal FLAG epitope.
Dominant negative
(DN) TLR1 and TLR6 expression vectors were constructed by subcloning
TIR (Toll and
interleukin 1 receptor) homology domain-deleted TLR1 and TLR6
fragments into pFLAG-CMV1
(pFLAG-dTLR1 and pFLAG-dTLR6). pHIV-LTR-luc, a mutant lacking the NF-
κB-binding
site (pHIV-LTRΔκB-luc), a mutant lacking the SP-1-binding site (pHIV-
LTRΔSP1-luc),
and a mutant lacking both NF-κB- and SP-1-binding sites (pHIV-
LTRΔκBSP1-luc) were
gifts from Dr Y. Koyanagi (Tohoku University Graduate School of
Medicine, Sendai,
Japan).40 The NF-κB Cis-Reporting System, containing pNF-kB-luc, a
plasmid in which
the luciferase reporter gene is fused to the NF-κB enhancer, was
purchased from
Stratagene (La Jolla, CA).
Transfection and luciferase assay
Transient transfection was performed by using FuGENE6 (Roche, Basel,
Switzerland),
according to the manufacturer's instructions. A total of 4 × 105 THP-1
cells,
or 1 × 105 293 T cells, were transfected with 0·1 µg of pFLAG-TLR2,
0·01 µg of pHIV-LTR-luc,
0·01 µg of the pRL-TK internal control plasmid (Promega, Madison, WI),
and DN TLRs
expressing plasmid, in 24-well plates. After 20 hr, transfected cells
were stimulated
with 1·0 µg/ml M. fermentans LAMPs or 0·5 µg/ml M. penetrans LAMPs.
After a further
24 hr of incubation, cells were lysed and assayed for luciferase
activity using
a Dual-Luciferase Reporter Assay System (Promega). Both firefly and
Renilla luciferase
activity were monitored using a Lumat LB9507 luminometer (Berthold,
Wildbad, Germany).
Normalized reporter activity is expressed as the firefly luciferase
value divided
by the Renilla luciferase value. Relative fold induction is calculated
as the normalized
reporter activity of the test samples divided by the unstimulated
samples.
Reverse-phase high-performance liquid chromatography (HPLC)
LAMPs were dissolved in 6 m guanidine hydrochloride, and 100 µg of
LAMPs were applied
on µBondasphere C18 300A (Waters, Milford, MA). Elution was carried
out using a
0–100% linear water/2-propanol gradient. The flow rate was 1·0 ml/min.
Each fraction
was dried in vacuo at room temperature and dissolved in 25 mm n-octyl-
β-gulucopyranoside.
Protein concentration was measured by using the Coomassie Protein
Assay Regent (Pierce).
Lipoprotein lipase treatment
Approxmately 100 ng/ml LPMf and LPMp, separated from M. fermentans and
M. penetrans
LAMPs, respectively, were treated with 100 µg/ml lipoprotein lipase
(Sigma) at 37°
for 2 hr. 293T cells transfected with 0·02 µg/ml pNF-kB-luc and 0·2 µg/
ml pFLAG-TLR2
were stimulated with 10 ng/ml of the lipoprotein lipase-treated LPMf
and LPMp. Luciferase
activity was measured as described above.
Top
Abstract
Introduction
Materials and methods
>Results
Discussion
References
Results
Activation of HIV LTR by LAMPs
LAMPs of M. penetrans have been reported to stimulate macrophages.
38,39 We therefore
initially examined whether LAMPs from M. fermentans and M. penetrans
can enhance
HIV replication in macrophages. To determine the enhancement of HIV
replication,
THP-1 cells were first transfected with a plasmid in which the
luciferase reporter
gene was fused to HIV LTR (pHIV-LTR-luc) and then were stimulated with
M. fermentans
and M. penetrans LAMPs. The level of luciferase expression was
enhanced by M. fermentans
or M. penetrans LAMPs in a dose-dependent manner (Fig. 1a). When 0·5
µg/ml M. penetrans
LAMPs was added, the luciferase expression was maximal and ≈ 12-fold
higher than
that of the unstimulated control. In contrast, the expression of THP-1
cells was
maximal when stimulated with 1·0 µg/ml M. fermentans LAMPs, being ≈ 10-
fold higher
compared with the control. These results indicate that LAMPs from M.
fermentans
and M. penetrans can enhance HIV replication.
Figure 1 Figure 1
Enhancement of long-terminal repeat (LTR) activation by lipid-
associated membrane
proteins (LAMPs) through nuclear factor-kappa B (NF-κB). (a) THP-1
cells were transfected
with 0·1 µg/ml pHIV-LTR-luc and 0·01 µg/ml (more ...)
Activation of HIV LTR through NF-κB
HIV LTR contains various binding sites of cellular transcription
factors, including
NF-κB, SP-1, AP2, TCF-1, USF-1 and Ets; in particular, NF-κB and SP-1
are thought
to be major transcription factors.41 To examine the roles of NF-κB and
SP-1 in the
activation of HIV LTR, pHIV-LTRΔκB-luc and pHIV-LTRΔSP1-luc (in which
the NF-κB-
and SP-1-binding sites, respectively, have been deleted from pHIV-LTR-
luc) were
prepared (Fig. 1b). When pHIV-LTRΔκB-luc-transfected THP-1 cells were
stimulated
with M. fermentans and M. penetrans LAMPs, the level of luciferase
expression was
lower than that of the control. In contrast, the level of luciferase
expression
was almost constant when pHIV-LTRΔSP1-luc was transfected. Moreover,
the deletion
of both NF-κB- and SP-1-binding sites (pHIV-LTRΔκBSP1-luc) resulted in
a decrease
of the expression level down to the level of the unstimulated control.
These results
indicate that NF-κB may be a major transcription factor induced by
LAMPs.
Inhibition of LTR activation by anti-TLR2 mAb
It was reported that a lipopeptide of M. fermentans– MALP-2 – can
activate NF-κB
through TLR2.28 We therefore examined whether the enhancement of HIV
LTR activation
with LAMPs is mediated through TLR2. Anti-human TLR2 mAb (ABM-8320)-
pretreated THP-1
cells were transfected with pHIV-LTR-luc, followed by stimulation with
M. fermentans
and M. penetrans LAMPs. Pretreatment with anti-TLR2 mAb decreased the
expression
level of luciferase, and control antibody (mouse IgG2a) had no effect
on luciferase
(Fig. 2a). These results indicate that the activation of HIV LTR by
LAMPs is TLR2-mediated.
Figure 2 Figure 2
Enhancement of long-terminal repeat (LTR) activation through Toll-like
receptor
2 (TLR2). (a) THP-1 cells were transfected with 0·1 µg/ml pHIV-LTR-luc
and 0·01
µg/ml pRL-TK. The cells were treated with anti-TLR2 monoclonal
(more ...)
Activation of HIV LTR through TLR2
To confirm whether the activation of NF-κB by LAMPs is mediated
through TLR2, we
constructed a TLR2 expression vector (pFLAG-TLR2). 293T cells were
transfected with
both pFLAG-TLR2 and pHIV-LTRΔSP1-luc. In this experiment, we used pHIV-
LTRΔSP1-luc
instead of pHIV-LTR-luc, because the binding site for SP-1 on the LTR
resulted in
a high level of luciferase activity (data not shown). When 293T cells
were transfected
with a high dose of pFLAG-TLR2 and then stimulated with M. fermentans
and M. penetrans
LAMPs, the levels of luciferase expression were augmented in a dose-
dependent manner
(Fig. 2b). In contrast, the level of luciferase expression was the
same as that
of the unstimulated control when 293T cells were transfected with the
empty vector
pFLAG-CMV1. This suggests that M. fermentans and M. penetrans LAMPs
activate HIV
LTR through TLR2.
Co-operation of TLR6 and TLR2 for LTR activation
Mouse TLR6 has been reported to recognize diacylated lipopeptides,
such as MALP-2,
co-operatively with TLR2.42 To investigate whether M. fermentans and
M. penetrans
LAMPs are also recognized by both TLR2 and TLR6 for the activation of
HIV LTR, we
constructed a plasmid encoding DN TLR6 (pFLAG-dTLR6). 293T cells were
transfected
with pFLAG-TLR2, pHIV-LTRΔSP1-luc and various concentrations of pFLAG-
dTLR6. Initially,
the effect of DN TLR6 on the expression of TLR2 was analysed by flow
cytometry.
The level of TLR2 expression was almost constant, irrespective of the
expression
of DN TLR6 or of DN TLR1 (data not shown). When the transfected cells
were stimulated
with M. fermentans LAMPs, the level of luciferase expression decreased
in a dose-dependent
manner (Fig. 3a). Upon transfection with 0·2 µg/ml pFLAG-dTLR6, the
expression level
decreased to a level similar to that of the unstimulated control. In
contrast, the
level of luciferase expression was almost constant when the
transfected 293T cells
were stimulated with M. penetrans LAMPs. These results suggest that
the LTR activation
by M. fermentans LAMPs is dependent on TLR2 and TLR6, but the
activation by M. penetrans
LAMPs is not dependent on TLR6. To further examine whether TLR6 alone
can mediate
the activation of LTR by LAMPs, 293T cells were transfected with a
TLR6 expression
vector (pFLAG-TLR6). Although the transfected cells were stimulated
with M. fermentans
and M. penetrans LAMPs, the level of luciferase expression was not
augmented (data
not shown). These results indicate that both TLR2 and TLR6 co-
operatively mediate
the LTR activation by M. fermentans LAMPs, but not by M. penetrans
LAMPs.
Figure 3 Figure 3
Cooperation of Toll-like receptor (TLR)1, TLR6 and TLR2 for long-
terminal repeat
(LTR) activation by lipid-associated membrane proteins (LAMPs). 293T
cells were
transfected with the indicated concentrations of pFLAG-dTLR6 (a) or
pFLAG-dTLR1
(b), 0·1 (more ...)
Co-operation of TLR1 and TLR2 for LTR activation
Triacylated bacterial lipopeptides, such as Pam3CSK4, were reported to
be recognized
by murine TLR1, in association with TLR2.43 The above results (Fig.
3a) suggest
that M. penetrans LAMPs might contain different active components from
M. fermentans
LAMPs and, like the triacylated lipopeptides, M. penetrans LAMPs might
be recognized
by TLR1 and TLR2. We therefore next determined whether M. fermentans
and M. penetrans
LAMPs are recognized by both TLR1 and TLR2 for the activation of HIV
LTR. To achieve
this, we transfected a plasmid encoding DN TLR1 (pFLAG-dTLR1) into
293T cells containing
both pFLAG-TLR2 and pHIV-LTRΔSP1-luc. When the transfected cells were
stimulated
with M. penetrans LAMPs, the level of luciferase expression was
decreased in a dose-dependent
manner (Fig. 3b). Unexpectedly, the level of luciferase expression of
the cells
stimulated with M. fermentans LAMPs was also decreased. Upon
transfection with 0·2
µg/ml pFLAG-dTLR1, the level of expression in both cells decreased
down to almost
control levels. These results suggest that the LTR activation by M.
fermentans and
M. penetrans LAMPs is dependent on both TLR1 and TLR2. To further
examine whether
TLR1 alone can mediate the activation of LTR by LAMPs, 293T cells were
transfected
with a TLR1 expression vector (pFLAG-TLR1). Like the TLR6 expression
in 293T cells,
as mentioned previously, the level of luciferase expression of the
transfected cells
was not augmented by stimulation with M. fermentans and M. penetrans
LAMPs (data
not shown). These results indicate that the co-operation of TLR1 and
TLR2 is required
for the LTR activation with M. fermentans and M. penetrans LAMPs.
Purification of active components of LAMPs
To purify the active components of LAMPs, M. penetrans and M.
fermentans LAMPs were
fractionated using reverse-phase HPLC with a linear gradient of
isopropanol. To
measure the activity of fractions to induce NF-κB, each fraction was
added to 293T
cells transfected with pFLAG-TLR2 and pNF-κB-luc. As shown in Fig.
4(a), the active
component of M. penetrans LAMPs (LPMp) was eluted by ≈ 97%
isopropanol, whereas
the active component of M. fermentans LAMPs (LPMf) was eluted by ≈ 80%
isopropanol
(Fig. 4b).
Figure 4 Figure 4
Isolation of active components of lipid-associated membrane proteins
(LAMPs). Mycoplasma
penetrans (a) and M. fermentans (b) LAMPS were dissolved in 6 m
guanidine hydrochloride,
and 100 µg of LAMPs was separated by reverse-phase high-performance
(more ...)
Analysis of active components of LAMPs
We next examined whether LPMp and LPMf separated from LAMPs are
recognized by TLR2
and TLR6, or TLR1 and TLR2. 293T cells transfected with pFLAG-TLR2,
pNF-κB-luc and
pFLAG-dTLR1 or pFLAG-dTLR6 were stimulated with LPMp, LPMf, Pam3CSK4
or MALP-2.
As reported previously by Takeuchi et al.,42,43 both DN TLR1 and DN
TLR6 suppressed
the activity of Pam3CSK4 and MALP-2, respectively, to induce NF-κB
(Fig. 5). When
the cells were stimulated with LPMp, the relative luciferase activity
was reduced
by the expression of DN TLR1 (Fig. 6), which is consistent with the
results obtained
using M. penetrans LAMPs (Fig. 3b). In contrast, the 293T cells
stimulated with
LPMf showed a relatively suppressed level of the luciferase activity
when DN TLR6,
but not DN TLR1, was expressed, inconsistent with the results that M.
fermentans
LAMPs was recognized by TLR1, TLR2 and TLR6 (Figs 3a and 5).
Figure 5 Figure 5
Toll-like receptor (TLR) usage of Mycoplasma penetrans lipid-
associated membrane
proteins (LPMp) and M. fermentans lipid-associated membrane proteins
(LPMf). 293T
cells transfected with 0·01 µg/ml pFLAG-TLR2, 0·01 µg/ml (more ...)
Figure 6 Figure 6
Lipoprotein lipase (LPL) treatment of Mycoplasma penetrans lipid-
associated membrane
proteins (LPMp) and M. fermentans lipid-associated membrane proteins
(LPMf). One
microgram of LPMp and LPMf was treated with 100 µg/ml LPL at 37° for
(more ...)
To analyse the chemical components of LPMf and LPMp, they were treated
with lipoprotein
lipase and proteinase K. Treatment with proteinase K failed to
decrease the activity
of LPMf and LPMp (data not shown), while lipoprotein lipase treatment
decreased
the ability to induce NF-κB (Fig. 6). These results suggest that lipid
moiety, but
not protein moiety, is required for activity.
Top
Abstract
Introduction
Materials and methods
Results
>Discussion
References
Discussion
In this study, we demonstrated that LAMPs from M. fermentans and M.
penetrans activated
the HIV LTR through NF-κB. Activation of the LTR by M. fermentans
LAMPs was TLR1-,
TLR2- and TLR6 dependent, while the activation by M. penetrans LAMPs
was TLR1- and
TLR2 dependent. The active components of M. fermentans and M.
penetrans LAMPs were
purified by reverse-phase HPLC. The purified lipoprotein from M.
penetrans LAMPs
(LPMp) was recognized by TLR1 and TLR2. Although the recognition of M.
fermentans
LAMPs involved TLR1, TLR2 and TLR6 (Fig. 3), the purified lipoprotein
from M. fermentans
LAMPs (LPMf) was recognized only by TLR2 and TLR6. These results
indicate that M.
fermentans LAMPs may contain several active components, in which one
component is
recognized by TLR2 and TLR6, and other components might be recognized
by TLR1 and
TLR2. Both LPMf and LPMp showed resistance to proteinase K treatment,
in agreement
with the previous report by Feng et al.39 In addition, the ability of
LPMf and LPMp
to induce NF-κB was reduced by treatment with lipoprotein lipase.
These results
suggest that the active components are attributable to lipid moieties.
MALP-2 constitutes
a lipopeptide, isolated from M. fermentans, which has been well
documented as an
activator of NF-κB.44,45 Subsequently, a 44-kDa membrane-bound
lipoprotein of M.
salivarium has been reported to induce TNF-α production in THP-1.46 To
our knowledge,
there still seems to be little information on the activity in innate
immune responses
of lipoproteins derived from mycoplasmas such as M. fermentans, M.
penetrans and
M. salivarium, although a variety of mycoplasma strains have been
shown to exhibit
diverse bioactivities in interaction with eukaryotic cells.47,48
In TLR-deficient mice, bacterial lipopeptides containing three acyl
chains were
reported to be recognized by TLR1 and TLR2,43 whereas MALP-2,
containing two acyl
chains, were recognized by TLR2 and TLR6.42 These findings, and our
results, suggest
that LPMp might be similar to lipoprotein(s) containing three acyl
chains. In contrast,
LPMf separated from M. fermentans in this study may possess components
comparable
to MALP-2, as MALP-2 is a lipopeptide derived from M. fermentans.
In mycoplasmas, acylated proteins are abundant cell-surface antigens,
and many putative
lipoprotein-encoding genes have been identified in the sequenced
mycoplasma genomes.49,50
It is, at present, controversial as to whether or not mycoplasmas have
triacylated
lipoprotein. Chemically identified lipoproteins from M. fermentans,
44M. hyorhinis,51M.
salivarium46 and M. gallisepticum52 are not N-acylated, nor has an N-
acyltransferase
gene been found in M. pneumoniae,53M. genitalium54 or M. penetrans55
genomes. To
date, the presence of proteins with N-acyltransferase activity has not
been clearly
established. However, the study on the ratio of N-amide and O-ester
bonds in M.
gallisepticum and M. mycoides may indicate the presence of diacylated
and triacylated
lipoproteins.56 The resistance to Edoman degradation of proteins from
M. mycoides
also indicates the presence of N-acylation.50 In this study, we found
that the lipoprotein
separated from M. penetrans induced NF-κB through TLR1 and TLR2.
Triacylated lipoproteins,
such as Pam3-CSK4, have been reported to be recognized by TLR1 and
TLR2,43 whereas
diacylated lipoproteins, such as MALP-2, have been shown to be
recognized by TLR2
and TLR6.42 Interestingly, synthetically triacylated MALP-2, N-
palamitoyl-MALP-2,
was not recognized by TLR6.57 These findings may indicate the
existence of triacylated
lipoproteins in mycoplasma species.
Our results indicate that the lipoproteins from M. fermentans and M.
penetrans can
activate NF-κB in HIV LTR, leading to the enhancement of HIV
replication. The activation
of NF-κB was also observed following stimulation with bacterial
components, including
LPS30 and peptidoglycan.24 We have previously reported that
glycolipids from Acholeplasma
laidlawii, binding to both HIV and macrophages, enhance HIV
replication.58,59 In
addition to the ability of lipoproteins to induce NF-κB, glycolipids
from mycoplasma
might, in concert, enhance the replication of HIV. We assume that
lipoproteins and
glycolipids residing in the mycoplasma membrane can efficiently attach
to the surface
of HIV-infected cells, as mycoplasmas are completely wall-less
bacteria.2 Moreover,
mycoplasmas contain various surface proteins that tend to show high-
frequency variation,
suggesting that mycoplasmas can escape from immune surveillance and
establish a
persistent infection.60 These findings suggest that mycoplasma, rather
than bacteria
with cell walls, might play an important role in the progression of
HIV infection.
We previously reported that a variety of mycoplasma strains can induce
TNF-α production
in mouse macrophages61 and THP-1 cells.62,63M. penetrans LAMPs have
been reported
to induce TNF-α production in mouse thioglycolate exudate peritoneal
macrophage
cells.39 Moreover, TNF-α has been shown to activate HIV LTR through NF-
κB.64 These
findings suggest that TNF-α produced by macrophages may activate NF-
κB. However,
we observed that anti-TNF-α mAb failed to inhibit the activation of
LTR by LAMPs
(data not shown). It is therefore unlikely that TNF-α produced by
LAMPs-stimulated
THP-1 cells may directly contribute to the activation of NF-κB.
In summary, we demonstrated that lipoproteins from the AIDS-associated
mycoplasmas,
M. fermentans and M. penetrans, can enhance HIV LTR activity in THP-1
cells through
NF-κB, and that this enhancement is dependent on TLRs. The enhancement
of the HIV
LTR activation induced by M. fermentans LAMPs was dependent on TLR1,
TLR2 and TLR6.
Interestingly, LPMf separated from M. fermentans LAMPs activated NF-κB
through TLR2
and TLR6, but not TLR1. In contrast, the enhancement of the NF-κB
activation induced
by M. penetrans LAMPs, as well as LPMp separated from M. penetrans
LAMPs, was dependent
on TLR1 and TLR2, but not TLR6. Clarifying the mechanisms by which
various bacteria,
including mycoplasma, enhance HIV replication may have therapeutic
values in preventing
the progression of AIDS during opportunistic infection.
Acknowledgments
We thank Dr Koyanagi for gifts of plasmids pHIV-LTR-luc, pHIV-LTRΔκB-
luc, pHIV-LTRΔSP1-luc
and pHIV-LTRΔκBSP1-luc. We also thank Dr Matsumoto for the gift of
MALP-2. This
work was supported, in part, by a grant from the Ishibashi Foundation.
kshe...@calea.org, fit...@gmail.com, patrick.f...@usdoj.gov,
model...@sbcglobal.net, jdr...@nejm.org, let...@courant.com,
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o...@po.state.ct.us, da...@davila-dilzer.com,
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Cc: fra...@ucia.gov, dr-ahma...@president.ir,
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billc...@gmail.com, thomas...@usdoj.gov, amcg...@rms-law.com,
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Subject: Pam3Cys (OspA) activates HIV replication
Date: Jul 21, 2008 8:45 AM
That's a considerable number of research-years and lives lost to the
lies
about LYMErix and Lyme Disease.
Imagine if Yale told the truth instead of blew these people off
deliberately:
http://www.actionlyme.org/SCHOEN_INSTRUCTING_DOCS_TO_BLOW_OFF_LYMERIX_INJUREES.htm
http://www.actionlyme.org/Bull_Lewis.htm
Your tax dollars at work.
http://www.actionlyme.org/BIOMARKERS2.htm
Paul Duray told these bastards about it in 1992.
KMDickson
http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=15312143
Immunology. 2004 September; 113(1): 121–129.
doi: 10.1111/j.1365-2567.2004.01937.x.
PMCID: PMC1782549
Copyright © 2004 Blackwell Publishing Ltd
Lipid-associated membrane proteins of Mycoplasma fermentans and M.
penetrans activate
human immunodeficiency virus long-terminal repeats through Toll-like
receptors
Takashi Shimizu, Yutaka Kida, and Koichi Kuwano
Department of Bacteriology, Kurume University School of Medicine,
Kurume, Japan
K. Kuwano, 67 Asahi-machi, Kurume, Fukuoka 830-0011, Japan. E-mail:
kuw...@med.kurume-u.ac.jp
Received April 21, 2004; Revised May 25, 2004; Accepted June 2, 2004.
Small right arrow pointing to: This article has been cited by other
articles in
PMC.
Top
>Abstract
Introduction
Materials and methods
Results
Discussion
References
Abstract
Mycoplasmas are known to enhance human immunodeficiency virus (HIV)
replication,
and mycoplasma-derived lipid extracts have been reported to activate
nuclear factor-κB
(NF-κB) through Toll-like receptors (TLRs). In this study, we examined
the involvement
of TLRs in the activation of HIV long-terminal repeats (LTR) by
mycoplasma and their
active components responsible for the TLR activation. Lipid-associated
membrane
proteins (LAMPs) from two species of mycoplasma (Mycoplasma fermentans
and M. penetrans)
that are associated with acquired immune-deficiency syndrome (AIDS),
were found
to activate HIV LTRs in a human monocytic cell line, THP-1. NF-κB
deletion from
the LTR resulted in inhibition of the activation. The LTR activation
by M. fermentans
LAMPs was inhibited by a dominant negative (DN) construct of TLR1 and
TLR6, whereas
HIV LTR activation by M. penetrans LAMPs was inhibited by DN TLR1, but
not by DN
TLR6. These results indicate that the activation of HIV LTRs by M.
fermentans and
M. penetrans LAMPs is dependent on NF-κB, and that the activation of
HIV LTR by
M. fermentans LAMPs is mediated through TLR1, TLR2 and TLR6. In
contrast, the LTR
activation by M. penetrans LAMPs is carried out through TLR1 and TLR2,
but not TLR6.
Subsequently, the active component of M. penetrans and M. fermentans
LAMPs was purified
by reverse-phase high-performance liquid chromatography (HPLC).
Interestingly, the
purified lipoprotein of M. penetrans LAMPs (LPMp) was able to activate
NF-κB through
TLR1 and TLR2. On the other hand, the activation of NF-κB by purified
lipoprotein
of M. fermentans LAMPs (LPMf) was mediated through TLR2 and TLR6, but
not TLR1.
Keywords: HIV, lipoprotein, mycoplasma, Toll-like receptor
Top
Abstract
>Introduction
Materials and methods
Results
Discussion
References
Introduction
Human immunodeficiency virus (HIV) is recognized as the aetiological
agent of acquired
immune-deficiency syndrome (AIDS). However, the progression of AIDS is
highly variable
in different individuals, and several factors, such as viral strains
or host factors,
have been attributed as the possible cause of such variations.
Infectious agents,
including various viruses, parasites and bacteria, are considered to
be cofactors
in the progression of AIDS.1 Mycoplasmas are wall-less parasitic Gram-
positive bacteria,
and the smallest organisms capable of self-replication.2Mycoplasma
fermentans and
M. penetrans have been isolated from the tissues and urine of patients
with AIDS3–5
and were shown to enhance the cytopathic effect of HIV-1 infection.6,7
In addition,
mycoplasmas and acholeplasmas have been reported to enhance HIV-1
replication in
vitro.8,9 Thus, mycoplasmas might be reasonable cofactor candidates in
the progression
of AIDS.
A nuclear transcription factor – nuclear factor-κB (NF-κB) – is
thought to play
a major role in the regulation of HIV-1 gene expression.10 Although
the HIV long-terminal
repeat (LTR) alone can serve as its own promoter, early mRNA
transcription appears
to rely primarily on the binding of cellular transcription factors,
including NF-κB,
to the LTR.11 The activation of cytoplasmic NF-κB by various
cytokines, including
interleukin (IL)-2, IL-6 and tumour necrosis factor-α (TNF-α), or
after infection
with other viruses, induces HIV replication.12–17 These findings
indicate increased
rates of HIV replication, probably through NF-κB-mediated regulation
of the HIV
LTR. TNF-α induced by mycoplasma had been thought to induce NF-κB and
HIV replication;
however, anti-TNF-α immunoglobulin failed to inhibit the enhancement
of HIV replication
by mycoplasma.18 Although activation of NF-κB has been implicated in
the mycoplasma-induced
enhancement of HIV replication, the receptor(s) and the pathways of
signal transduction
via NF-κB have not been clearly defined.
Recently, it has been reported that Toll-like receptors (TLRs) are
pattern-recognition
receptors in the innate immune system and play important roles in
early innate recognition
and inflammatory responses by the host to microbial challenges.19
Among nine TLR
family members reported, TLR2, 4, 5 and 9 have been implicated in the
recognition
of different bacterial components. Pepidoglycan, lipoarabinomannan,
zymosan and
lipoproteins from various micro-organisms are recognized by TLR2.20–28
On the other
hand, lipopolysaccharide (LPS), bacterial flagellin and bacterial DNA
are recognized
by TLR4, TLR5 and TLR9, respectively.29–33 These TLR family members
have been shown
to activate NF-κB via IL-1R-associated signal molecules, including
myeloid differentiation
protein (MyD88), IL-1R-activated kinase (IRAK), TNFR-associated factor
6 (TRAF6),
and NF-κB-inducing kinase (NIK).34 However, the precise mechanisms by
which mycoplasma
activate HIV LTR have not been fully clarified.
In this study, we examined the involvement of TLRs in the activation
of HIV LTRs
by mycoplasmas and their active components responsible for the TLR
activation. We
observed that lipid-associated membrane proteins (LAMPs) from the AIDS-
associated
mycoplasmas, M. penetrans and M. fermentans, activated the HIV LTR in
a human monocytic
cell line (THP-1) through NF-κB. Activation of the HIV LTR by LAMPs
from M. fermentans
(referred to as M. fermentans LAMPs hereafter) was apparently
dependent on TLR1,
TLR2 and TLR6. In contrast, activation of the HIV LTR by LAMPs from M.
penetrans
(referred to as M. penetrans LAMPs hereafter) was dependent on TLR1
and TLR2, but
not on TLR6. Furthermore, the active components of M. penetrans and M.
fermentans
LAMPs were purified by reverse-phase HPLC. The activity of purified
lipoprotein
from M. penetrans LAMPs (LPMp) to induce NF-κB was dependent on TLR1
and TLR2. On
the other hand, the activity of purified lipoprotein from M.
fermentans LAMPs (LPMf)
was dependent on TLR2 and TLR6, but not on TLR1.
Top
Abstract
Introduction
>Materials and methods
Results
Discussion
References
Materials and methods
Cells
Cells of a human monocytic cell line, THP-1, were cultured in
RPMI-1640 containing
10% fetal calf serum (FCS; Mitsubishi Chemical, Tokyo, Japan), 2 mm l-
glutamine,
100 U/ml penicillin G and 100 µg/ml streptomycin. Cells of a human
kidney cell line,
293T, were cultured in Dulbecco's modified Eagle's minimal essential
medium
(DMEM) containing 10% FCS, 2 mm l-glutamine, 100 U/ml penicillin G and
100 µg/ml
streptomycin.
Antibodies
The mouse anti-human TLR2 monoclonal antibodies (mAbs) ABM-8320 and
IMG-416 were
obtained from Cascade Bioscience (Winchester, MA) and Imgenex (San
Diego, CA).35
Normal mouse immunoglobulin G (IgG)2a was purchased from PharMingen
(San Diego,
CA).
Pathogen-associated molecular patterns (PAMPs)
(S)-[2,3-Bis(palmitoyloxy)-(2-RS)-propyl]-N-palmitoyl-(R)-Cys-(S)-Ser-
(S)-Lys4-OH.3HCl
(Pam3CSK4) was purchased from Calbiochem (Darmstadt, Germany). M.
fermentans macrophage-activating
lipopeptide 2 (MALP-2) was kindly provided by Dr M. Matsumoto (Osaka
Medical Center
for Cancer and Cardiovascular Diseases, Osaka, Japan).36,37
Preparation of LAMPs from M. fermentans and M. penetrans
M. fermentans and M. penetrans were cultured in PPLO medium and SP-4
medium, respectively,
to the start of stationary phase, and then pelleted by centrifugation
for 10 min
at 12 000 g. Preparation of LAMPs was performed as described
previously by Feng
et al.38,39 Briefly, a mycoplasma pellet was suspended in Tris-
buffered saline (TBS)
(50 mm Tris, 0·15 m NaCl, pH 8·0) containing 1 mm EDTA (TBSE),
solubilized by adding
TX-114 to a final concentration of 2% and incubated at 4° for 1 hr.
The lysate was
incubated at 37° for 10 min prior to phase separation. After
centrifugation at 10
000 g for 20 min, the upper aqueous phase was removed and replaced
with the same
volume of TBSE. The procedure of phase separation was repeated twice.
The final
TX-114 phase was resuspended in TBSE to the original volume, 2·5
volumes of ethanol
were added to precipitate membrane components and the phase was
incubated at −20°
overnight. After centrifugation, the pellet was suspended in phosphate-
buffered
saline (PBS) followed by sonication for 30 seconds at output 5
(Sonifier cell disruptor
200; Branson, Danbury, CT). The protein concentration of the
suspension was measured
by using the Coomassie Protein Assay Regent (Pierce, Rockford, IL).
Expression vectors
To prepare TLR1, TLR2 and TLR6 expression vectors (pFLAG-TLR1, pFLAG-
TLR2, and pFLAG-TLR6,
respectively), the coding regions of TLR1, TLR2 and TLR6, minus the
respective N-terminal
signal sequences, were amplified by polymerase chain reaction (PCR)
from a cDNA
of THP-1 and cloned into the expression vector pFLAG-CMV1 (Sigma, St
Louis, MO),
in which a preprotrypsin leader precedes an N-terminal FLAG epitope.
Dominant negative
(DN) TLR1 and TLR6 expression vectors were constructed by subcloning
TIR (Toll and
interleukin 1 receptor) homology domain-deleted TLR1 and TLR6
fragments into pFLAG-CMV1
(pFLAG-dTLR1 and pFLAG-dTLR6). pHIV-LTR-luc, a mutant lacking the NF-
κB-binding
site (pHIV-LTRΔκB-luc), a mutant lacking the SP-1-binding site (pHIV-
LTRΔSP1-luc),
and a mutant lacking both NF-κB- and SP-1-binding sites (pHIV-
LTRΔκBSP1-luc) were
gifts from Dr Y. Koyanagi (Tohoku University Graduate School of
Medicine, Sendai,
Japan).40 The NF-κB Cis-Reporting System, containing pNF-kB-luc, a
plasmid in which
the luciferase reporter gene is fused to the NF-κB enhancer, was
purchased from
Stratagene (La Jolla, CA).
Transfection and luciferase assay
Transient transfection was performed by using FuGENE6 (Roche, Basel,
Switzerland),
according to the manufacturer's instructions. A total of 4 × 105 THP-1
cells,
or 1 × 105 293 T cells, were transfected with 0·1 µg of pFLAG-TLR2,
0·01 µg of pHIV-LTR-luc,
0·01 µg of the pRL-TK internal control plasmid (Promega, Madison, WI),
and DN TLRs
expressing plasmid, in 24-well plates. After 20 hr, transfected cells
were stimulated
with 1·0 µg/ml M. fermentans LAMPs or 0·5 µg/ml M. penetrans LAMPs.
After a further
24 hr of incubation, cells were lysed and assayed for luciferase
activity using
a Dual-Luciferase Reporter Assay System (Promega). Both firefly and
Renilla luciferase
activity were monitored using a Lumat LB9507 luminometer (Berthold,
Wildbad, Germany).
Normalized reporter activity is expressed as the firefly luciferase
value divided
by the Renilla luciferase value. Relative fold induction is calculated
as the normalized
reporter activity of the test samples divided by the unstimulated
samples.
Reverse-phase high-performance liquid chromatography (HPLC)
LAMPs were dissolved in 6 m guanidine hydrochloride, and 100 µg of
LAMPs were applied
on µBondasphere C18 300A (Waters, Milford, MA). Elution was carried
out using a
0–100% linear water/2-propanol gradient. The flow rate was 1·0 ml/min.
Each fraction
was dried in vacuo at room temperature and dissolved in 25 mm n-octyl-
β-gulucopyranoside.
Protein concentration was measured by using the Coomassie Protein
Assay Regent (Pierce).
Lipoprotein lipase treatment
Approxmately 100 ng/ml LPMf and LPMp, separated from M. fermentans and
M. penetrans
LAMPs, respectively, were treated with 100 µg/ml lipoprotein lipase
(Sigma) at 37°
for 2 hr. 293T cells transfected with 0·02 µg/ml pNF-kB-luc and 0·2 µg/
ml pFLAG-TLR2
were stimulated with 10 ng/ml of the lipoprotein lipase-treated LPMf
and LPMp. Luciferase
activity was measured as described above.
Top
Abstract
Introduction
Materials and methods
>Results
Discussion
References
Results
Activation of HIV LTR by LAMPs
LAMPs of M. penetrans have been reported to stimulate macrophages.
38,39 We therefore
initially examined whether LAMPs from M. fermentans and M. penetrans
can enhance
HIV replication in macrophages. To determine the enhancement of HIV
replication,
THP-1 cells were first transfected with a plasmid in which the
luciferase reporter
gene was fused to HIV LTR (pHIV-LTR-luc) and then were stimulated with
M. fermentans
and M. penetrans LAMPs. The level of luciferase expression was
enhanced by M. fermentans
or M. penetrans LAMPs in a dose-dependent manner (Fig. 1a). When 0·5
µg/ml M. penetrans
LAMPs was added, the luciferase expression was maximal and ≈ 12-fold
higher than
that of the unstimulated control. In contrast, the expression of THP-1
cells was
maximal when stimulated with 1·0 µg/ml M. fermentans LAMPs, being ≈ 10-
fold higher
compared with the control. These results indicate that LAMPs from M.
fermentans
and M. penetrans can enhance HIV replication.
Figure 1 Figure 1
Enhancement of long-terminal repeat (LTR) activation by lipid-
associated membrane
proteins (LAMPs) through nuclear factor-kappa B (NF-κB). (a) THP-1
cells were transfected
with 0·1 µg/ml pHIV-LTR-luc and 0·01 µg/ml (more ...)
Activation of HIV LTR through NF-κB
HIV LTR contains various binding sites of cellular transcription
factors, including
NF-κB, SP-1, AP2, TCF-1, USF-1 and Ets; in particular, NF-κB and SP-1
are thought
to be major transcription factors.41 To examine the roles of NF-κB and
SP-1 in the
activation of HIV LTR, pHIV-LTRΔκB-luc and pHIV-LTRΔSP1-luc (in which
the NF-κB-
and SP-1-binding sites, respectively, have been deleted from pHIV-LTR-
luc) were
prepared (Fig. 1b). When pHIV-LTRΔκB-luc-transfected THP-1 cells were
stimulated
with M. fermentans and M. penetrans LAMPs, the level of luciferase
expression was
lower than that of the control. In contrast, the level of luciferase
expression
was almost constant when pHIV-LTRΔSP1-luc was transfected. Moreover,
the deletion
of both NF-κB- and SP-1-binding sites (pHIV-LTRΔκBSP1-luc) resulted in
a decrease
of the expression level down to the level of the unstimulated control.
These results
indicate that NF-κB may be a major transcription factor induced by
LAMPs.
Inhibition of LTR activation by anti-TLR2 mAb
It was reported that a lipopeptide of M. fermentans– MALP-2 – can
activate NF-κB
through TLR2.28 We therefore examined whether the enhancement of HIV
LTR activation
with LAMPs is mediated through TLR2. Anti-human TLR2 mAb (ABM-8320)-
pretreated THP-1
cells were transfected with pHIV-LTR-luc, followed by stimulation with
M. fermentans
and M. penetrans LAMPs. Pretreatment with anti-TLR2 mAb decreased the
expression
level of luciferase, and control antibody (mouse IgG2a) had no effect
on luciferase
(Fig. 2a). These results indicate that the activation of HIV LTR by
LAMPs is TLR2-mediated.
Figure 2 Figure 2
Enhancement of long-terminal repeat (LTR) activation through Toll-like
receptor
2 (TLR2). (a) THP-1 cells were transfected with 0·1 µg/ml pHIV-LTR-luc
and 0·01
µg/ml pRL-TK. The cells were treated with anti-TLR2 monoclonal
(more ...)
Activation of HIV LTR through TLR2
To confirm whether the activation of NF-κB by LAMPs is mediated
through TLR2, we
constructed a TLR2 expression vector (pFLAG-TLR2). 293T cells were
transfected with
both pFLAG-TLR2 and pHIV-LTRΔSP1-luc. In this experiment, we used pHIV-
LTRΔSP1-luc
instead of pHIV-LTR-luc, because the binding site for SP-1 on the LTR
resulted in
a high level of luciferase activity (data not shown). When 293T cells
were transfected
with a high dose of pFLAG-TLR2 and then stimulated with M. fermentans
and M. penetrans
LAMPs, the levels of luciferase expression were augmented in a dose-
dependent manner
(Fig. 2b). In contrast, the level of luciferase expression was the
same as that
of the unstimulated control when 293T cells were transfected with the
empty vector
pFLAG-CMV1. This suggests that M. fermentans and M. penetrans LAMPs
activate HIV
LTR through TLR2.
Co-operation of TLR6 and TLR2 for LTR activation
Mouse TLR6 has been reported to recognize diacylated lipopeptides,
such as MALP-2,
co-operatively with TLR2.42 To investigate whether M. fermentans and
M. penetrans
LAMPs are also recognized by both TLR2 and TLR6 for the activation of
HIV LTR, we
constructed a plasmid encoding DN TLR6 (pFLAG-dTLR6). 293T cells were
transfected
with pFLAG-TLR2, pHIV-LTRΔSP1-luc and various concentrations of pFLAG-
dTLR6. Initially,
the effect of DN TLR6 on the expression of TLR2 was analysed by flow
cytometry.
The level of TLR2 expression was almost constant, irrespective of the
expression
of DN TLR6 or of DN TLR1 (data not shown). When the transfected cells
were stimulated
with M. fermentans LAMPs, the level of luciferase expression decreased
in a dose-dependent
manner (Fig. 3a). Upon transfection with 0·2 µg/ml pFLAG-dTLR6, the
expression level
decreased to a level similar to that of the unstimulated control. In
contrast, the
level of luciferase expression was almost constant when the
transfected 293T cells
were stimulated with M. penetrans LAMPs. These results suggest that
the LTR activation
by M. fermentans LAMPs is dependent on TLR2 and TLR6, but the
activation by M. penetrans
LAMPs is not dependent on TLR6. To further examine whether TLR6 alone
can mediate
the activation of LTR by LAMPs, 293T cells were transfected with a
TLR6 expression
vector (pFLAG-TLR6). Although the transfected cells were stimulated
with M. fermentans
and M. penetrans LAMPs, the level of luciferase expression was not
augmented (data
not shown). These results indicate that both TLR2 and TLR6 co-
operatively mediate
the LTR activation by M. fermentans LAMPs, but not by M. penetrans
LAMPs.
Figure 3 Figure 3
Cooperation of Toll-like receptor (TLR)1, TLR6 and TLR2 for long-
terminal repeat
(LTR) activation by lipid-associated membrane proteins (LAMPs). 293T
cells were
transfected with the indicated concentrations of pFLAG-dTLR6 (a) or
pFLAG-dTLR1
(b), 0·1 (more ...)
Co-operation of TLR1 and TLR2 for LTR activation
Triacylated bacterial lipopeptides, such as Pam3CSK4, were reported to
be recognized
by murine TLR1, in association with TLR2.43 The above results (Fig.
3a) suggest
that M. penetrans LAMPs might contain different active components from
M. fermentans
LAMPs and, like the triacylated lipopeptides, M. penetrans LAMPs might
be recognized
by TLR1 and TLR2. We therefore next determined whether M. fermentans
and M. penetrans
LAMPs are recognized by both TLR1 and TLR2 for the activation of HIV
LTR. To achieve
this, we transfected a plasmid encoding DN TLR1 (pFLAG-dTLR1) into
293T cells containing
both pFLAG-TLR2 and pHIV-LTRΔSP1-luc. When the transfected cells were
stimulated
with M. penetrans LAMPs, the level of luciferase expression was
decreased in a dose-dependent
manner (Fig. 3b). Unexpectedly, the level of luciferase expression of
the cells
stimulated with M. fermentans LAMPs was also decreased. Upon
transfection with 0·2
µg/ml pFLAG-dTLR1, the level of expression in both cells decreased
down to almost
control levels. These results suggest that the LTR activation by M.
fermentans and
M. penetrans LAMPs is dependent on both TLR1 and TLR2. To further
examine whether
TLR1 alone can mediate the activation of LTR by LAMPs, 293T cells were
transfected
with a TLR1 expression vector (pFLAG-TLR1). Like the TLR6 expression
in 293T cells,
as mentioned previously, the level of luciferase expression of the
transfected cells
was not augmented by stimulation with M. fermentans and M. penetrans
LAMPs (data
not shown). These results indicate that the co-operation of TLR1 and
TLR2 is required
for the LTR activation with M. fermentans and M. penetrans LAMPs.
Purification of active components of LAMPs
To purify the active components of LAMPs, M. penetrans and M.
fermentans LAMPs were
fractionated using reverse-phase HPLC with a linear gradient of
isopropanol. To
measure the activity of fractions to induce NF-κB, each fraction was
added to 293T
cells transfected with pFLAG-TLR2 and pNF-κB-luc. As shown in Fig.
4(a), the active
component of M. penetrans LAMPs (LPMp) was eluted by ≈ 97%
isopropanol, whereas
the active component of M. fermentans LAMPs (LPMf) was eluted by ≈ 80%
isopropanol
(Fig. 4b).
Figure 4 Figure 4
Isolation of active components of lipid-associated membrane proteins
(LAMPs). Mycoplasma
penetrans (a) and M. fermentans (b) LAMPS were dissolved in 6 m
guanidine hydrochloride,
and 100 µg of LAMPs was separated by reverse-phase high-performance
(more ...)
Analysis of active components of LAMPs
We next examined whether LPMp and LPMf separated from LAMPs are
recognized by TLR2
and TLR6, or TLR1 and TLR2. 293T cells transfected with pFLAG-TLR2,
pNF-κB-luc and
pFLAG-dTLR1 or pFLAG-dTLR6 were stimulated with LPMp, LPMf, Pam3CSK4
or MALP-2.
As reported previously by Takeuchi et al.,42,43 both DN TLR1 and DN
TLR6 suppressed
the activity of Pam3CSK4 and MALP-2, respectively, to induce NF-κB
(Fig. 5). When
the cells were stimulated with LPMp, the relative luciferase activity
was reduced
by the expression of DN TLR1 (Fig. 6), which is consistent with the
results obtained
using M. penetrans LAMPs (Fig. 3b). In contrast, the 293T cells
stimulated with
LPMf showed a relatively suppressed level of the luciferase activity
when DN TLR6,
but not DN TLR1, was expressed, inconsistent with the results that M.
fermentans
LAMPs was recognized by TLR1, TLR2 and TLR6 (Figs 3a and 5).
Figure 5 Figure 5
Toll-like receptor (TLR) usage of Mycoplasma penetrans lipid-
associated membrane
proteins (LPMp) and M. fermentans lipid-associated membrane proteins
(LPMf). 293T
cells transfected with 0·01 µg/ml pFLAG-TLR2, 0·01 µg/ml (more ...)
Figure 6 Figure 6
Lipoprotein lipase (LPL) treatment of Mycoplasma penetrans lipid-
associated membrane
proteins (LPMp) and M. fermentans lipid-associated membrane proteins
(LPMf). One
microgram of LPMp and LPMf was treated with 100 µg/ml LPL at 37° for
(more ...)
To analyse the chemical components of LPMf and LPMp, they were treated
with lipoprotein
lipase and proteinase K. Treatment with proteinase K failed to
decrease the activity
of LPMf and LPMp (data not shown), while lipoprotein lipase treatment
decreased
the ability to induce NF-κB (Fig. 6). These results suggest that lipid
moiety, but
not protein moiety, is required for activity.
Top
Abstract
Introduction
Materials and methods
Results
>Discussion
References
Discussion
In this study, we demonstrated that LAMPs from M. fermentans and M.
penetrans activated
the HIV LTR through NF-κB. Activation of the LTR by M. fermentans
LAMPs was TLR1-,
TLR2- and TLR6 dependent, while the activation by M. penetrans LAMPs
was TLR1- and
TLR2 dependent. The active components of M. fermentans and M.
penetrans LAMPs were
purified by reverse-phase HPLC. The purified lipoprotein from M.
penetrans LAMPs
(LPMp) was recognized by TLR1 and TLR2. Although the recognition of M.
fermentans
LAMPs involved TLR1, TLR2 and TLR6 (Fig. 3), the purified lipoprotein
from M. fermentans
LAMPs (LPMf) was recognized only by TLR2 and TLR6. These results
indicate that M.
fermentans LAMPs may contain several active components, in which one
component is
recognized by TLR2 and TLR6, and other components might be recognized
by TLR1 and
TLR2. Both LPMf and LPMp showed resistance to proteinase K treatment,
in agreement
with the previous report by Feng et al.39 In addition, the ability of
LPMf and LPMp
to induce NF-κB was reduced by treatment with lipoprotein lipase.
These results
suggest that the active components are attributable to lipid moieties.
MALP-2 constitutes
a lipopeptide, isolated from M. fermentans, which has been well
documented as an
activator of NF-κB.44,45 Subsequently, a 44-kDa membrane-bound
lipoprotein of M.
salivarium has been reported to induce TNF-α production in THP-1.46 To
our knowledge,
there still seems to be little information on the activity in innate
immune responses
of lipoproteins derived from mycoplasmas such as M. fermentans, M.
penetrans and
M. salivarium, although a variety of mycoplasma strains have been
shown to exhibit
diverse bioactivities in interaction with eukaryotic cells.47,48
In TLR-deficient mice, bacterial lipopeptides containing three acyl
chains were
reported to be recognized by TLR1 and TLR2,43 whereas MALP-2,
containing two acyl
chains, were recognized by TLR2 and TLR6.42 These findings, and our
results, suggest
that LPMp might be similar to lipoprotein(s) containing three acyl
chains. In contrast,
LPMf separated from M. fermentans in this study may possess components
comparable
to MALP-2, as MALP-2 is a lipopeptide derived from M. fermentans.
In mycoplasmas, acylated proteins are abundant cell-surface antigens,
and many putative
lipoprotein-encoding genes have been identified in the sequenced
mycoplasma genomes.49,50
It is, at present, controversial as to whether or not mycoplasmas have
triacylated
lipoprotein. Chemically identified lipoproteins from M. fermentans,
44M. hyorhinis,51M.
salivarium46 and M. gallisepticum52 are not N-acylated, nor has an N-
acyltransferase
gene been found in M. pneumoniae,53M. genitalium54 or M. penetrans55
genomes. To
date, the presence of proteins with N-acyltransferase activity has not
been clearly
established. However, the study on the ratio of N-amide and O-ester
bonds in M.
gallisepticum and M. mycoides may indicate the presence of diacylated
and triacylated
lipoproteins.56 The resistance to Edoman degradation of proteins from
M. mycoides
also indicates the presence of N-acylation.50 In this study, we found
that the lipoprotein
separated from M. penetrans induced NF-κB through TLR1 and TLR2.
Triacylated lipoproteins,
such as Pam3-CSK4, have been reported to be recognized by TLR1 and
TLR2,43 whereas
diacylated lipoproteins, such as MALP-2, have been shown to be
recognized by TLR2
and TLR6.42 Interestingly, synthetically triacylated MALP-2, N-
palamitoyl-MALP-2,
was not recognized by TLR6.57 These findings may indicate the
existence of triacylated
lipoproteins in mycoplasma species.
Our results indicate that the lipoproteins from M. fermentans and M.
penetrans can
activate NF-κB in HIV LTR, leading to the enhancement of HIV
replication. The activation
of NF-κB was also observed following stimulation with bacterial
components, including
LPS30 and peptidoglycan.24 We have previously reported that
glycolipids from Acholeplasma
laidlawii, binding to both HIV and macrophages, enhance HIV
replication.58,59 In
addition to the ability of lipoproteins to induce NF-κB, glycolipids
from mycoplasma
might, in concert, enhance the replication of HIV. We assume that
lipoproteins and
glycolipids residing in the mycoplasma membrane can efficiently attach
to the surface
of HIV-infected cells, as mycoplasmas are completely wall-less
bacteria.2 Moreover,
mycoplasmas contain various surface proteins that tend to show high-
frequency variation,
suggesting that mycoplasmas can escape from immune surveillance and
establish a
persistent infection.60 These findings suggest that mycoplasma, rather
than bacteria
with cell walls, might play an important role in the progression of
HIV infection.
We previously reported that a variety of mycoplasma strains can induce
TNF-α production
in mouse macrophages61 and THP-1 cells.62,63M. penetrans LAMPs have
been reported
to induce TNF-α production in mouse thioglycolate exudate peritoneal
macrophage
cells.39 Moreover, TNF-α has been shown to activate HIV LTR through NF-
κB.64 These
findings suggest that TNF-α produced by macrophages may activate NF-
κB. However,
we observed that anti-TNF-α mAb failed to inhibit the activation of
LTR by LAMPs
(data not shown). It is therefore unlikely that TNF-α produced by
LAMPs-stimulated
THP-1 cells may directly contribute to the activation of NF-κB.
In summary, we demonstrated that lipoproteins from the AIDS-associated
mycoplasmas,
M. fermentans and M. penetrans, can enhance HIV LTR activity in THP-1
cells through
NF-κB, and that this enhancement is dependent on TLRs. The enhancement
of the HIV
LTR activation induced by M. fermentans LAMPs was dependent on TLR1,
TLR2 and TLR6.
Interestingly, LPMf separated from M. fermentans LAMPs activated NF-κB
through TLR2
and TLR6, but not TLR1. In contrast, the enhancement of the NF-κB
activation induced
by M. penetrans LAMPs, as well as LPMp separated from M. penetrans
LAMPs, was dependent
on TLR1 and TLR2, but not TLR6. Clarifying the mechanisms by which
various bacteria,
including mycoplasma, enhance HIV replication may have therapeutic
values in preventing
the progression of AIDS during opportunistic infection.
Acknowledgments
We thank Dr Koyanagi for gifts of plasmids pHIV-LTR-luc, pHIV-LTRΔκB-
luc, pHIV-LTRΔSP1-luc
and pHIV-LTRΔκBSP1-luc. We also thank Dr Matsumoto for the gift of
MALP-2. This
work was supported, in part, by a grant from the Ishibashi Foundation.
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