Purification of antimicrobial factors from human
Li Ming1,2, Pan Xiaoling2, Li Yan3, Wang Lili2, Wu Qi2, Yu Xiyong1, Wang Boyao2
and Huang Ning2,4
1Research Center of Medical Science, Guangdong Provincial People’s Hospital, Guangzhou 510080, People’s Republic of China;
2Research Unit of Infection and Immunity, West China Medical Center, Sichuan University, Chengdu 610041, People’s Republic
of China;3College of Basic Medicine, Guangzhou University of Traditional Chinese Medicine, Guangzhou 510405,
People’s Republic of China
4Correspondence address. Tel: þ86-28-85503159; Fax: þ86-28-85501549; E-mail: email@example.com
BACKGROUND: The aim of this study was to separate bactericidal proteins from healthy female cervical mucus.
METHODS: Cervical mucus was collected and dissolved in 1% acetic acid. The antimicrobial activity of acid-
solubleextracts was detected by gel overlayassay againstEscherichia coli ATCC43827.The extracts showedconsider-
able amount of antibacterial activity with a clearly visible band. The bactericidal band was purified by reverse-phase
high performance liquid chromatography and the antibacterial activity of the eluate was examined using radial diffu-
sion assay. RESULTS: Two antimicrobial proteins were purified and were further characterized by Tricine sodium
dodecyl sulphate-polyacrylamide gel electrophoresis, N-terminal sequencing and matrix-assisted laser desorption ion-
ization time-of-flight mass spectrometry. The proteins were identified as high-mobility group nucleosomal-binding
domain 2 (HMG N2) and secretory leukocyte peptidase inhibitor (SLPI). SLPI is an antimicrobial peptide already
known in the cervical mucus while HMG N2 in the cervical mucus had not been previously reported. The expression
of HMG N2 mRNAwas detected in Hela cells andcervical epithelial cells by RT-PCR. Slit hybridization showed abun-
dant amounts of the HMG N2 protein in the cervical mucus. CONCLUSIONS: These results suggest that the
expression of HMG N2 and SLPI in the healthy female cervical mucus may be relevant to their immune surveillance
and defense against potential pathogens in human reproductive system.
Keywords: innate immune; antimicrobial protein; cervical mucus; high-mobility group nucleosomal-binding domain 2;
secretory leukocyte peptidase inhibitor
In recent years, antimicrobial peptides and proteins, with the
capacity to kill a broad spectrum of microorganisms, have
become recognized as key mediators of the innate immunity
in many organisms (Ganz, 2005; Silphaduang et al., 2006).
Successful human implantation and pregnancy are reliant on
the prevention of genital tract infections that can compromise
both fertility and the fetus itself. The molecules of the innate
immune system, present at mucosal surfaces, are likely to be
crucial in the limitation of such infections (King et al.,
2002). The female reproductive tract is an important site of
antibacterial peptide production. Several antimicrobial pep-
tides have been detected in the reproductive tissues including
lysozyme (Eggert-Kruse et al., 2000), human beta defensin
(HBD) 1–4 (King et al., 2002; King et al., 2003), human
defensin (HD) 5 (Svinarich et al., 1997) and secretory leuko-
cyte peptidase inhibitor (SLPI) (King et al., 2000; King
et al., 2002). Our group has also found that HBD 1 and HBD
2 are located in female genital tract and pregnancy-related
tissues (Feng et al., 2003). The presence of these molecules
suggests a role in the protection of the reproductive tract
Cervical mucus is a viscous, gel-like fluid located between
microbe-rich vagina and the normally sterile uterine cavity,
which suggests a host defense function other than a physical
barrier preventing ascending infections (Hein et al., 2001).
The cervical mucus is well known as a multifactorially deter-
mined filtering system, but not much is known about its anti-
microbial activity (Eggert-Kruse et al., 2000). Even though
antibacterial activity of cervical mucus had been found pre-
viously, none of the antimicrobial peptides, except SLPI
(Moriyama et al., 1999) and lysozyme (Eggert-Kruse et al.,
2000), had been purified from the cervical mucus. The aims
of the present investigation were to identify novel antibacter-
ial molecules and characterize these molecules as potentially
important factorsintheinnateimmunity ofhuman
# The Author 2007. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.
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Human Reproduction Vol.22, No.7 pp. 1810–1815, 2007
Advance Access publication on May 18, 2007
by guest on June 7, 2013
reproductive tract. Our results showed that cervical mucus
exhibited antibacterial activity in vitro and two antimicrobial
factors (including one previously in reported factor) were sep-
arated from it.
Materials and Methods
Collection of cervical mucus
The Regional Research Ethics Committee approved the project and
consent was obtained from each volunteer. The women (n ¼ 8) were
healthy, with a median age of 26 (range 24–35) years. The parity
was 1, and the specimens were taken at least one year after delivery.
All the women had regular menstrual cycles (27–31 days). None of
them had taken any antibiotics for at least 4 weeks or hormones for
3 months prior to biopsy collection. At least 5 days before material
collection, the volunteers refrained from sexual intercourse. They pre-
sented with a normal-appearance cervix and without pathological cer-
vical discharge or symptoms of genital tract infection. All the samples
were obtained at 3–4 days after menstruation by aspiration with a 1 ml
syringe from the cervix.
The samples were dissolved in 1% acetic acid, homogenized and
centrifuged at 12 000g for 30 min at 48C. The supernatant was col-
lected and dialysed against water overnight at 48C. The samples
were then lyophilized and re-dissolved in 0.01% acetic acid. Protein
concentration was measured by bicinchoninic acid protein assay
(Pierce Biotechnology Inc, USA). All samples were stored at
2808C for future use.
Gel overlay antibacterial assay
Gel overlay assay was performed according to the protocol of Lehrer
et al. (1991). Briefly, Escherichia coli ATCC 43827 cells were grown
overnight in Luria–Bertani broth and centrifuged at 12 000g for
10 min at 48C. The pellet was washed twice using 20 mM phosphate-
buffered saline (PBS) (pH 7.2). The bacterial concentration was
adjusted to 1 ? 106CFU/ml by using a colorimeter (Bio-rad Inc,
USA). Then the bacteria were mixed with underlay gel solution kept
molten at 438C to have the final concentration of bacteria at 1 ? 105
CFU/ml. The underlay gel was composed of 1% agar, 0.3%
soybean extract and 100 mM phosphate buffer (pH 6.8). The
mixture was immediately poured into an integrid Petri dish (Fisher
scientific, USA) to form a uniform layer of ?1 mm deep.
Crude extracts (30 mg) of cervical mucus were subjected to non-
(AU-PAGE) in duplicate. After electrophoresis, the gel was cut into
two identical halves. One half was washed by rinsing in 0.01 M PB
(pH 7.2) for 10 min to decrease the acetic acid and urea content.
Then the PAGE gel was placed on the top of the underlay gel. The
dish was incubated for 3 h at 378C to allow the peptides to diffuse
into the underlay gel from the PAGE gel. The PAGE gel was
removed and replaced by an overlay gel containing 1% agar and 6%
soybean extract. The chimeric gel was incubated overnight at 378C,
to enable the surviving bacteria to form microcolonies. Clear colony-
free regions were seen where the growth of bacteria was suppressed by
protein bands with antibacterial activity. The second half of the gel, an
exact replica of the first half, was stored in 0.01% acetic acid in MilliQ
water, and was used for the excision of antimicrobial zones.
Purification of the antimicrobial proteins
Peptides were excised from the AU-PAGE replica and purified. The
purification included preparative AU-PAGE elution and reverse-phase
high performance liquid chromatography (RP-HPLC). The prepara-
tive AU-PAGE elution was referenced from the protocol of Harwig
et al. (1993). The eluates of preparative AU-PAGE were lyophilized,
reconstituted in 0.01% acetic acid and then injected into RP-HPLC
with a 4.6 ? 250 mm Vydac C18 column (Agilent Inc, USA).
Bound materials were eluted with a linear gradient of acetonitrile
(ACN) in acidified water [0.1% trifluoroacetic acid (TFA) in water
to 60% ACN with 0.1% TFA] for 60 min at a flow rate of 1.0 ml
per minute. The elution profile was monitored at 214 nm. All fractions
were collected, lyophilized, reconstituted with 0.01% acetic acid and
stored at 2808C for further tests of their antimicrobial activities.
Radial diffusion antimicrobial assay
The underlay gel containing bacteria was prepared exactly as
described above for the gel overlay assay. A series of wells, each
3 mm in diameter, were punched into the solidified underlay gel.
Around 5 ml of samples (1 mg) or positive control (lysozyme,
10 mg) was added into designated wells. The dish was incubated at
378C for 3 h to allow the peptide to diffuse into the underlay gel
from the wells. A molten overlay gel was poured, and the dish was
incubated overnight at 378C until zones of inhibition were visible.
The clear zones around the holes indicating no growth of bacteria
were seen when the samples contained antibacterial effectors.
Electrophoresis and electrotransfer to membrane
Tricine sodium dodecyl sulphate-polyacrylamide gel electrophoresis
(Tricine SDS-PAGE) for low-molecular weight proteins was per-
formed as described by Schagger et al. (1987). The SDS gel had a sep-
arating gel of 16.5% acrylamide with a 10% acrylamide spacer gel and
4% stacking gel. The gel was subjected to electrophoresis for 3 h at
80 V and stained by silver staining. For the samples used in N-terminal
sequencing or mass spectrometry analysis, the peptides were electro-
transfered from gel to membrane before staining. Electrotransfer was
performed at 20 V for 40 min. ProBlott polyvinylidene difluoride
(PVDF) membranes (0.45 mm, Invitrogen Inc, USA) stained with
Coomassie Brillant Blue R-250 (Sigma, USA) were used for sequence
analysis while nitrocellulose membranes (0.22 mm, Invitrogen Inc)
stained with Amido Black were used for mass spectrometry.
The amino acid sequencing was done at Peking University, Beijing,
China. N-terminal amino acid sequencing of the antimicrobial factor
was performed by sequential Edman degradation method using
Procise 477 automatic sequence analysis system (Applied Biosystems,
USA) according to standard protocols of manufacturer.
Protein identification by mass spectrometry analysis was performed
at Chengdu Branch of Chinese Academy of Sciences, Chengdu,
China. The molecular weight of the isolated antimicrobial factor
was determined by a mass spectrometric method using a matrix-
assisted laser desorption/ionization time-of-flight mass spectrometer
(MALDI-TOF-MS) instrument (Bruker Daltonics, Germany).
Primary epithelial cell culture
Cervical tissues (n ¼ 5) were obtained from women undergoing total
hysterectomy because of hysteromyoma or endometriosis. Consent
was obtained from each patient and the investigation was approved
by the Regional Research Ethics Committee. Cervical tissues were
checked by pathologic examination. Samples without pathological
changes were used in following procedure. Cervical tissues were
digested by 0.1% protease and 0.1% DNAase at 48C for 36 h. The
supernatant was collected and then centrifuged at 1000g for 10 min.
The pellet was washed three times with PBS, for 5 min each time.
Antimicrobial factors in human cervical mucus
by guest on June 7, 2013
The primary cervical epithelial cells were cultured according to the
protocol of Gorodeski et al. (1994) with some modification. Briefly,
the epithelial cells were cultured in 153 culture medium (Sigma) sup-
plemented with Insulin (5 g/ml, Sigma), hydrocortisone (0.5 g/ml,
Sigma), transferrin (5 g/ml, Sigma), epidermal growth factor
(10 ng/ml, Sigma), epinephrine (7.5 g/ml, Sigma), bovine pituitary
extracts (20 ng/ml, Sigma), penicillin (50 mg/ml, Sigma) and strepto-
mycin (50 mg/ml, Sigma). There is evidence that these factors are
likely to beinvolved in modulationof cell proliferation, differentiation
and maturation (Stanley et al., 1984; Chopra et al., 1991; Gorodeski
et al., 1994; Myohanen et al., 2001). Cells were grown to near conflu-
ence (7–9 days) in a 6-well plate.
Hela cells (Shanghai Branch of Chinese Academy of Sciences, Shang-
hai) was grown in RPMI 1640 (Gibcol BRL, USA) supplemented with
10% fetal calf serum (Gibcol BRL), penicillin (50 mg/ml, Sigma) and
streptomycin (50 mg/ml, Sigma).
Nucleic acid extraction and RT-PCR analyses
Total RNA was extracted with Trizol reagent (Invitrogen Inc) from the
cervical epithelial cells and Hela cells. RNA samples were then treated
with RNase-free DNase I (Promega, USA) to remove cellular DNA.
RNA concentrations and qualities were verified by optical density
measurement and agarose gels electrophoresis. The Superscript II
cDNA kit (Invitrogen Inc) was utilized to synthesize cDNA. A PCR
reaction was performed with sense primer (50AACGGATCC
TTTGCCCTGCTG) and antisense primer (50CCAGGATCCGG
CACACACTAG). PCR reaction mixtures were made to contain
cDNA (3 ml), rTaq DNA polymerase (0.025 IU/ml; Takara, Japan),
MgCl2(2 mM), dNTPs (all at 200 mM) and specific sense and anti-
sense primers (300 nM; Takara). A no template control (containing
water) was included. The PCR reactions were run on Eppendorf Mas-
tercycler 5331 (Eppendorf, German) and the thermocycling conditions
were as follows: initial denaturation for 5 min at 958C; followed by
30 cycles of 958C for 45 s, 528C for 30 s and 728C for 30 s, and then
ments were performed at least three times. The products were analysed
by electrophoresis through 2% agarose gel and DNA sequencing.
Slit hybridization of human cervical mucus
The expression of recombinant high-mobility group nucleosomal-
binding domain 2 (HMG N2) protein and preparation of rabbit
anti-HMG N2 polyclone antibody were performed by our laboratory
as described elsewhere (Xiong et al., 2005). Slit hybridization was
carried out using the Hoefer Dot Blot system (GE, USA) following
the protocol provided by the manufacture. Briefly, 5 ml of extract pro-
teins from human cervical mucus (15 mg) were dotted on PVDF mem-
brane and dried before proceeding to the blocking step. Subsequently,
the membrane was blocked with blocking buffer (1% nonfat dry milk,
20 mM Tris–HCl, 500 mM NaCl, 0.1% Tween-20, pH 7.5) for 2 h
with gentle shaking at room temperature. After incubation the mem-
brane was washed with washing buffer (20 mM Tris–HCl, 500 mM
NaCl, 0.1% Tween-20, pH 7.5) for 10 min with gentle agitation.
The membrane was then incubated overnight at 48C with rabbit
anti-HMG N2 antibody diluted to 1:1000 in the blocking buffer.
After overnight incubation the membrane was rinsed twice with
washing buffer before being incubated with goat anti-rabbit
IgG-horseradish peroxidase second antibody (Dako, Denmark),
which was diluted to 1:5000 in blocking buffer, at room temperature
for 1 h. To remove the excess antibody, the membrane was washed
twice with washing buffer. Finally, color signals of the membrane
was obtained following incubation with the 3, 3-diaminobenzidine
(Dako) for 10 min, and washed with distilled water to stop the reac-
tion. Recombinant HMG N2 protein was used as positive control
and bovine serum albumin (BSA), as negative control.
We found that the cervical mucus was a vicious hydrogel
weighing between 0.15 and 0.30 g. Examined with paper
strips, the median pH of the cervical mucus was 7.2, with a
range of 6.4–8.0. The cervical index was determined (Insler
et al., 1972) and the score of cervical mucus was no greater
than 2 in all donors. The mucus samples contained no blood
contamination. The number of leukocytes was ,1 (mean of
10 visual fields) per high power field (?400).
Antibacterial activity of human cervical mucus
The antibacterial activity of acid-soluble proteins from human
cervical mucus was tested by the gel overlay assay (Fig. 1).
Lysozyme, a known antibacterial peptide, was used as positive
control. The electrophoresed antimicrobial proteins were sepa-
rated on a polyacrylamide gel and the molecules were trans-
ferred to a bacterial-inoculated agarose gel (the underlay gel
described above) by blotting. After overnight incubation,
bacteria-free regions were clearly visible. Because proteins
with similar ionic charge cannot be separated by AU-PAGE,
the band containing antibacterial activity maybe includes
several proteins. Therefore the band was cut off from the
PAGE gel for further separation and purification.
Purification of antimicrobial proteins
The active band was separated by RP-HPLC into .10 fractions
(Fig. 2A). Radial diffusion assay was used to assess the
Figure 1: Gel overlay assay of antibacterial activity of human cervi-
cal mucus acid-soluble extracts against E. coli ATCC 43827
(A) AU-PAGE electrophoresis of acid-soluble extracts of cervical
mucus and lysozyme. (B) Evaluation of the microbicidal activity of
lysozyme and cervical mucus. A major clear zone shown in the
agarose indicated the bacterial growth inhibition by corresponding
Ming et al.
by guest on June 7, 2013
21 min) and fraction 26 (eluted at 26 min), presented high levels
of antimicrobial activity against E. coli ATCC 43827 (Fig. 2B).
Electrophoresis of selected chromatography fractions
The fractions 21 and 26 shown in Fig. 2B were analysed by
Tricine SDS-PAGE electrophoresis and stained by silver stain-
ing (Fig. 2C). Fractions 21 and 26 displayed only one protein
band each, at ?14 and 12 kDa, respectively (Fig. 2C).
Identification of proteins
The first 10 amino acid residues of the N-terminal sequence of
fraction 21 were P-K-R-K-A-E-G-D-A-K, which was exactly
corresponding to the N-terminal sequence of the human
HMG N2 through the BLAST program assay (http://www.
ncbi.nlm.nih.gov/BLAST/). Molecular mass of fraction 21
were determined using MALDI-TOF-MS. The molecular
mass of fraction 21 was 9263.62 Da (Fig. 3), in a good agree-
ment with the molecular mass of HMG N2. It suggested that
fraction 21 should be as entire molecules of HMG N2, an anti-
bacterial effector not previously detected in reproductive tract.
The molecular mass of fraction 21 determined by MALDI-
TOF-MS was lower than the relative mass for fraction 21 indi-
cated by mobility on Tricine SDS-PADE (Fig. 2C), which was
14 kDa. It may be explained by the aberrant behavior of HMG
protein in these conditions as reported by Einck et al. (1985).
The first 10 amino acid residues of the N-terminal sequence
of fraction 26 were S-G-K-S-F-K-A-G-V-C, which was identi-
cal tothe N-terminal sequence of SLPI. The calculated molecu-
lar weight of SLPI is 11.7 kDa, in exact agreement with the
molecular mass observed in the Tricine SDS-PAGE. It has
been reported that SLPI is a constitutively expressed natural
antibiotics in female reproductive tract (King et al., 2000,
2002). Therefore fraction 26 was identified as SLPI.
HMG N2 expression in cervical epithelial cells
Employing RT-PCR, we revealed the presence of HMG N2
mRNA in the cervical epithelial cells and Hela cells. Agarose
gel electrophoresis of PCR products showed that there was a
specific band at about 270 bp (Fig. 4), in accordance with our
expectation. The results of DNA sequencing confirmed that
the fragment was 270 bp in length, and identical to HMG N2.
Slit hybridization of human cervical mucus
A slit hybridization assay was performed to prove the hypo-
thesis that HMG N2 is present in human cervical mucus.
Immunostaining was observed in the cervical mucus and posi-
tive control, whereas no staining was visible in negative
control. The results revealed the presence of the HMG N2
protein in the cervical mucus of healthy women (Fig. 5).
The antimicrobial activity of cervical mucus had been noted,
but its content of antimicrobial peptides has not been systema-
tically analysed. This investigation focused specifically on the
separation and identification of new antibacterial effectors
Figure 2: Purification of antibacterial peptides (A) Fractionation of
proteins contained in the band with antibacterial activity by
RP-HPLC. The band with antibacterial activity was eluated by pre-
parative AU-PAGE electrophoresis. The eluates were subjected to
RP-HPLC on a 4.6 ? 250 mm Vydac C18 column and eluted with
linear gradient of ACN in acidified water (0.1% TFA in water to
60% ACN with 0.1% TFA) for 60 min at a flow rate of 1.0 ml per
minute. Arrows indicated the antibacterial fractions 21 and 26.
(B) Evaluation of the microbicidal activity of RP-HPLC fractions.
Eluate was collected per minute. Agar radial diffusion assay indicated
the fractions 21 and 26 contained antibacterial activities against E. coli
ATCC 43827. Well 1 was filled with lysozyme, well 2 with fraction
21, well 3 with fraction 26 and well 4 with 0.01% acetic acid as nega-
tive control. (C) Tricine SDS-PAGE electrophoresis of chromato-
graphy fractions 21 and 26. M, molecular weight of standard; Lane
1, fraction 21 and Lane 2, fraction 26
Figure 3: Molecular weight determination of antimicrobial protein
isolated from cervical mucus
Figure 4: Analysis by RT-PCR reveals the expression of HMG N2
mRNA in Hela cells and human cervical epithelial cells. Expression
of HMG N2 mRNA was seen in cervical epithelial cells (lanes A
and B), as well as in Hela cells (lane C). In the negative control
(lane D) no RNA was added to the PCR reaction. M, molecular
weight of standard. These results were representative of three indepen-
Antimicrobial factors in human cervical mucus
by guest on June 7, 2013
from healthy female cervical mucus. In the present study, two
antibacterial peptides, HMG N2 and SLPI were identified.
The HMG protein family consists of six proteins and each of
these proteins seems to have a distinct type of function in the
nucleus (Bustin, 1999). However, it is well known that peptides
of the HMG protein family have additional functions. For
example, the HMG box chromosomal protein 1 (HMG B1) is
widely known as a nuclear DNA-binding protein. Now, there
is growing body of evidence demonstrating that HMG B1 pos-
sesses potent bactericidal activity (Zetterstrom et al., 2006).
HMG N2 gene is located in chromosome 1p36.1, and it con-
tains six exons, with an extremely high GC content and an
‘Hpa II tiny fragment’ island. Until now, the biological role
of HMG N2 has not been defined fully. A variety of experi-
ments have shown that HMG N2 is associated with chromatin
subunits (Hock et al., 1998). Furthermore, the abnormal gene
or protein expression of HMG N2 is related to some diseases
such as neoplasms (Spieker et al., 2000) and autoimmune dis-
eases (Ayer et al., 1994). The significance of HMG N2 in the
host innate immune against infections was reported by our lab-
oratory (Feng et al., 2005). A previous study showed that HMG
N2 was an antimicrobial molecule with potent activity against
E. coli, Pseudomonas aeruginosa and Candida albicans. An
amphipathic a-helical domain was found in the HMG N2
protein, which was proved to be essential for its antimicrobial
activity. The present study demonstrated HMG N2 located in
female cervical mucus, which was further confirmed by slit
hybridization. To our knowledge, this is the first report describ-
ing detection of HMG N2 presented in human cervical mucus.
The mRNA of HMG N2 was detected in Hela cells and primary
cervical epithelial cells using RT-PCR, which indicated that
HMG N2 was expressed in cervical epithelial cell. Thus,
HMG N2 could be regarded as antibacterial effector released
by the cervical epithelial cells to combat invading bacteria in
SLPI has been previously detected in female reproductive
system, including the cervix (Helmig et al., 1995), endome-
trium (King et al., 2000, 2002), first trimester deciduas (King
et al., 2000), term deciduas (Hiemstra et al., 1996) and
amnion fluid (Denison et al., 1999). SLPI was thought to func-
tion mainly as a serine protease inhibitor. Now, SLPI has been
found to have antibacterial, antiviral and antifungal effects
(Zhang et al., 2001). Our observation of SLPI in cervical
mucus confirmed the previous reports. It should be noted that
SLPI still had the ability to protect antimicrobial peptides
from excessive proteolysis and inactivation by inflammatory
proteases (Tomee et al., 1998).
The identification of antimicrobial factors in cervical mucus
was based on the method of Bourgeon et al. (2004). The advan-
tage of this method was that it involved the direct identification
ofmolecules responsible for the observed antimicrobial effects.
It is of great potential value for the identification of novel anti-
microbial effectors in immunoprivileged biological fluids and
tissues. Several antibacterial molecules have been identified
using this method from different tissues including the male
and female reproductive tract (Bourgeon et al., 2004; Mak
et al., 2004; Silphaduang et al., 2006). As cervical mucus is
a vicious gel-like fluid, solubilization is necessary to ensure a
homogeneous mixture. Sonification was proved to be an effi-
cient method for dispersion of cervical mucus (Eggert-Kruse
et al., 2000). It was used in all samples. Enzymatic treatment
was not used because of its proteolytic activity. The antibacter-
ial molecules studied to date are all cationic peptides with a
high isoelectric point (Reddy et al., 2004), therefore, more
soluble in a mild acid solution. Acid extraction of tissue was
performed to enhance the solubility of the antibacterial proteins.
In fact, the antibacterial activity of cervical mucus extracted by
acid solution was more potent than when extracted by water
solution. Similar results were obtained by King et al. (2000).
Moreover, no other antimicrobial peptides, such as defensins
and lysozyme,were detected inthe cervical mucus by using our
experimental method. We postulated that the healthy female
used in this study did not constitutively produce such mole-
cules, or these factors were expressed at a low level that our
method could not detect it. It has been shown that defensins
are inducible antimicrobial peptides in epithelial cells of endo-
metrial (King et al., 2002) and other multiple organs (Bals
et al., 1999; Milner et al., 2003). The possible reason that lyso-
zyme was not detected in our study might be that the approach
we used was different from Eggert-Kruse’s (2000). A previous
paper reported that HNP1-3, b-defensin, lactoferrin and lyso-
zyme are present in cervical mucus plugs from pregnant
females (Hein et al., 2001). It is considered that the antibacter-
ial activity is augmented in pregnancy because of the different
endocrine status. For example, the concentrations of lysozyme
in cervical mucus plugs are about twice as high as in cervical
mucus. Therefore, healthy females may have to rely on consti-
tutively expressed immune surveillance factors, such as HMG
N2 and SLPI, for a basal level of immune defense, while others
are inducible allowing maximal antimicrobial activity during
infection or pregnancy.
Ascending infections are major cause of infertility and
adverse pregnancy outcome. It is reported that up to 20% of
preterm births are associated with uterine infections (Romero
et al., 1989). The main role for HMG N2 and SLPI in cervical
mucus is likely to be in the prevention of ascending infections.
Even though the vagina is polymicrobially colonized and is
known to be ecological niches, with a specific transient and
resident flora, the uterine cavity is usually sterile (Eggert-Kruse
et al., 2000). One explanation for this might be the presence of
local antibacterial factors in the cervical mucus. HMG N2 and
Figure 5: Slit hybridization of cervical mucus with anti-HMG N2
antibody. Slit B was filled with recombinant HMG N2 protein, slit
C with cervical mucus and slit A with BSA as negative control
Ming et al.
by guest on June 7, 2013
SLPI were shown to have antimicrobial activity, hence they
may constitute cervix surveillance system designed to protect
the uterus from infections.
Our study only identified the antimicrobial molecules of
cervical mucus from healthy females. The contents of cervical
mucus are influenced by several variables such as endocrine
status, sexual intercourse and infections. More samples from
healthy and infectious donors with different endocrine status’
and different menstruation stages should be collected for
In conclusion, we find that female cervical mucus harbors
components with bactericidal activity. These components
have been identified as HMG N2 and SLPI. The presence of
HMG N2 and SLPI in healthy female cervical mucus may be
relevant to their immune surveillance and defense against
potential pathogens in the human reproductive system.
This work was supported by Chinese Medical Board of New York
(No 96-681) and National Natural Science Foundation of China
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Submitted on January 29, 2007; resubmitted on April 8, 2007; accepted on
April 17, 2007
Antimicrobial factors in human cervical mucus
by guest on June 7, 2013